J Endocrinol Invest (2014) 37:189–194 DOI 10.1007/s40618-013-0036-7
ORIGINAL ARTICLE
Frequency and effect on serum TSH of phosphodiesterase 8B (PDE8B) gene polymorphisms in patients with sporadic nonautoimmune subclinical hypothyroidism P. Agretti • G. De Marco • C. Di Cosmo • B. Bagattini • E. Ferrarini • L. Montanelli P. Vitti • M. Tonacchera
•
Received: 10 July 2013 / Accepted: 16 November 2013 / Published online: 9 January 2014 Ó Italian Society of Endocrinology (SIE) 2013
Abstract Background Nonautoimmune subclinical hypothyroidism (NSH) is characterized by elevated serum TSH in presence of normal thyroid hormone levels and absence of antithyroid antibodies. As result of a genomic-wide study, a strong association between three polymorphic variants in intron 1 of human PDE8B gene (rs4704397, rs6885099, rs2046045) and serum TSH has been reported in euthyroid subjects. Aim The aim of this study was to evaluate frequency and effects on serum TSH of PDE8B gene polymorphisms in patients with sporadic NSH and verify if differences in serum TSH levels are associated to these polymorphic variants. Subjects and methods A total of 58 Italian selected patients affected by NSH, with elevated serum TSH, normal FT3 and FT4 and without TSHr gene mutations, were subjected to genotyping for specific single nucleotide polymorphism of PDE8B gene. Results In all patients, the integrity of TSH receptor gene was attested. The ancestral allele associated with increased serum TSH was present in 42/58 patients (72.4 %) for rs4704397, in 42/58 patients (72.4 %) for rs6885099 and in 44/58 patients (75.9 %) for rs2046045. However, similar
P. Agretti G. De Marco C. Di Cosmo B. Bagattini E. Ferrarini L. Montanelli P. Vitti M. Tonacchera Dipartimento di Medicina Clinica e Sperimentale, Sezione di Endocrinologia, Universita` di Pisa, Azienda Ospedaliero Universitaria Pisana, Pisa, Italy M. Tonacchera (&) Dipartimento di Medicina Clinica e Sperimentale, Sezione di Endocrinologia, Universita` di Pisa, Via Paradisa 2, 56124 Pisa, Italy e-mail: [email protected]
values of serum TSH were detected in patients with minor or major allele for each polymorphism. Conclusions A prevalence of the minor allele of PDE8B gene polymorphism associated with elevated serum levels of TSH was demonstrated in patients affected by sporadic NSH; however, significant differences in circulating TSH in patients with minor or major alleles for each polymorphism were not identified demonstrating the lack of association between the polymorphisms and serum TSH levels in these patients. Keywords PDE8B gene Single nucleotide polymorphisms Nonautoimmune subclinical hypothyroidism Circulating TSH
Introduction Subclinical hypothyroidism (SH) defines a condition characterized by elevated serum TSH associated with normal circulating FT4 and FT3 [1]. The most common etiology of SH in western countries is chronic autoimmune thyroiditis that may progress toward clinically overt hypothyroidism [2]. SH due to chronic autoimmune thyroiditis must be distinguished from the rare condition of thyroid resistance to TSH (nonautoimmune subclinical hypothyroidism, NSH), a congenital syndrome of variable hyposensitivity to a biologically active TSH molecule [3]. The defect is characterized by elevated serum TSH and normal to very low serum levels of thyroid hormones in the presence of a hypoplastic or normally sized gland in the proper position in the neck. Depending on the degree of impairment of TSHr function, subjects can present with euthyroid hyperthyrotropinemia at one extreme of the spectrum, or severe hypothyroidism at the other [3, 4].
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Germline loss of function mutations of the TSH receptor gene has been described in the case of partial or complete TSH resistance [5–8]. In the last years, variants of different genes have been associated with susceptibility of normal population to alteration of thyroid function [9, 10]. In particular, three single nucleotide polymorphisms (SNPs) in PDE8B gene (rs4704397, rs6885099, rs2046045) have been found to be strongly associated with altered serum TSH concentrations in general population [11]. By genotyping 362,129 SNPs in 4,300 Sardinians, the authors identified the strongest association between alleles of rs4704397 and circulating serum TSH levels; each additional copy of the minor A allele was associated with an increase of 0.13 lU/ml in TSH. A strong association has also been demonstrated for serum TSH levels and SNPs rs6885099 and rs2046045 [11]. A meta-analysis by Taylor et al. [12] 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. The polymorphism rs4704397 of PDE8B gene was also demonstrated to be associated with SH in pregnancy [13] and with serum TSH levels in obese children [14]. On the other hand obesity, as known, positively correlates with elevated serum concentration of TSH [15, 16]. The PDE8B gene (PDE8B, MIM ?603390) codifies for a thyroid-specific phosphodiesterase, which catalyzes the hydrolysis of cAMP in thyroid and thereby regulates cAMP levels in the gland. From the foregoing, it would affect the production of thyroid hormones and TSH release from pituitary modulating thyroid physiology [11]. PDE8B polymorphisms may reduce cAMP in the thyroid leading to a decreased thyroid stimulatory response to TSH and lower thyroid hormone production. This is likely to result in a higher TSH level required to maintain FT4 and FT3 in the normal range due to the negative feedback [13]. In this paper, we aimed to assess the frequency of the three PDE8B gene polymorphisms showing the strongest association with circulating TSH levels in a group of patients affected by sporadic NSH and to verify if different TSH serum levels were associated with different polymorphic variants of PDE8B in patients with idiopathic NSH.
Materials and methods Study group Our study group consisted of 58 patients affected by NSH. Thirty-five females and 23 males with a mean age of 27.1 ± 14.1 years (ranging from 4 to 73 years, with the same normal range of TSH) were included in the study. In
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all 58 subjects, serum thyroid hormone levels were within the normal range (FT4 mean 10.1 ± 2.1 pg/ml with normal values of 7.0–17.0 pg/ml, FT3 mean 3.9 ± 0.8 pg/ml with normal values of 2.7–5.7 pg/ml) and serum TSH level was elevated (mean TSH 7.6 ± 5.7 lU/ml ranging from 3.6 to 38.0 lU/ml, with a normal range of 0.4–3.4 lU/ml). Serial dilutions of the TSH immunoreactivity were performed in all cases and were always parallel to the standard curve (data not shown). Test for antithyroperoxidase (TPOAb), antithyroglobulin (TgAb) and antiTSH-receptor (TRAb) antibodies gave negative results. Ultrasound of the thyroid showed a normal gland in the proper position in the neck with a normoechogenic pattern. After intravenous TRH challenge, the surge of TSH was proportional to basal TSH values and was followed by an adequate production of FT4 and FT3, suggesting a normal bioactivity of the TSH molecule (data not shown). In all 58 patients, no thyroid defect was identified in the parents and relatives investigated. Subjects of pediatric age received L-T4 therapy, which normalized serum TSH level. Adult patients with NSH were not treated with L-T4 replacement therapy, and serum thyroid hormone and TSH levels were checked once a year. After a follow-up of 3 years, serum TSH concentrations were unchanged with values similar to those recorded at the first observation. None of the subjects developed in vitro or in vivo features of thyroid autoimmunity. Subjects with signs of atherosclerotic cardiovascular disease or with other chronic diseases that could affect endothelial function such as obesity and diabetes were not included in the study group. All 58 patients were comparable with respect to lipid profile and their resting electrocardiograms were normal. In Table 1, the anthropometric and clinical characteristics of the patients are represented. The study was approved by the local ethical committee, and informed consent was obtained from all subjects. Laboratory evaluation of thyroid function Serum FT3 and FT4 were measured with a chemiluminescent method (Vitro System, Ortho-Clinical Diagnostics, Rochester, NY, USA). Thyrotropin (TSH) was assessed by Table 1 Anthropometric and clinical features of patients affected by sporadic or familial nonautoimmune subclinical hypothyroidism Age (years)
27.1 ± 14.1
Weight (kg)
58.9 ± 15.1
Height (cm)
157.3 ± 22.3
BMI TSH (lU/ml)
23.4 ± 2.1 7.6 ± 5.7
FT3 (pg/ml)
3.9 ± 0.8
FT4 (pg/ml)
10.1 ± 2.1
Average values and standard deviations are reported
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ultrasensitive commercial chemiluminescent method (Immulite 2000; Diagnostic Products, Los Angeles, CA, USA). TPOAb and TgAb were measured using a two-step immunoenzymatic assay (AIA-Pack TgAb and TPOAb; Tosoh, Tokyo, Japan). Serum Tg was measured using an immunometric chemiluminescence assay (Immulite 2000; Diagnostic Products, Los Angeles, CA, USA). TSH receptor (TSHr) antibodies were measured using a commercial radioreceptor assay (TRAK assay, B.R.A.H.M.S., Berlin, Germany).
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population, and in our case permitted to assess differences between the three genotypes of polymorphisms rs4704397, rs6885099 and rs2046045. The critical value for the Kruskal–Wallis H test comes from the Chi-square table with k - 1 degrees of freedom, where k is the number of groups being tested. The computed value must be larger than the critical value in order to reject the null hypothesis.
Results Sequence determination and genotyping Genomic DNA was extracted from peripheral lymphocytes of the patients using standard procedures [17]. All coding exons of the TSHr gene were PCR amplified and sequenced using previously described methods [6], and the sequencing products were analyzed on a 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Fifty nanograms of genomic DNA were used to genotype patients for specific SNPs of PDE8B gene by predeveloped TaqMan SNP genotyping assay (Applied Biosystems, Foster City, CA, USA). Allele-specific probes were labeled with the fluorescent dyes VIC and FAM, respectively. PCR was carried out in a total reaction volume of 20 ll with the following amplification protocol: denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 1 min. To obtain a high confidence level, the manufacturer recommended to run for each assay four no-template controls, four allele 1 controls and four allele 2 controls. The ABI PRIM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) was used to detect and measure the fluorescence signals generated from the PCR amplification during an endpoint plate read. After the plate read was completed, SDS software calculated the fluorescence measurements made during the plate read and plotted fluorescence values based on signals from each well; in this way, the genotype of each sample and control was determined. A positive signal indicates a perfect match between the probe and the tested DNA, thus identifying the allele types. Statistical analysis Circulating serum levels of TSH for the study group were not normally distributed, while FT3 and FT4 values were approximately normally distributed. Descriptive statistics for the study group are presented as median and interquartile range, and nonparametric analysis was carried out using the Kruskall–Wallis H test [13]. This method permits to assess differences between median values of different groups where there is not a normal distribution of
We selected 58 patients with sporadic NSH who did not harbor TSHr mutations. In five patients, the polymorphic variant P52T of the TSHr was present, while in one patient the D36H and P52T polymorphic variants of the TSHr were simultaneously present. All genetic variants of TSHr gene were identified at the heterozygous state (data not shown). The haplotype associated with increased serum TSH level is A for rs4704397, G for rs6885099 and G for rs2046045 and each one corresponds to the ancestral allele. The ancestral allele A of rs4704397, the polymorphism of PDE8B gene which shows the strongest association to elevated circulating levels of serum TSH, was present in 42/58 subjects corresponding to about 72 %; this allele was present in the homozygous state (AA) in 15/58 subjects (25.8 %) and in the heterozygous state (AG) in 27/58 subjects (46.6 %). The ancestral allele G of rs6885099 PDE8B gene polymorphism was present in 42/58 subjects corresponding to about 72 %; this allele was present in the homozygous state (GG) in 14/58 subjects (24.1 %) and in the heterozygous state (GA) in 28/58 subjects (48.3 %). The ancestral allele G of rs2046045 PDE8B gene polymorphism was present in 44/58 subjects corresponding to about 76 %; this allele was present in the homozygous state (GG) in 16/58 subjects (27.6 %) and in the heterozygous state (GT) in 28/58 subjects (48.3 %) (Table 2).
Table 2 Frequency of PDE8B polymorphisms in patients affected by sporadic or familial nonautoimmune subclinical hypothyroidism
rs4704397 (A/G) rs6885099 (G/A) rs2046045 (G/T)
Allele 1
Allele 1/2
Allele 2
Total allele 1
15/58
27/58
16/58
42/58
25.8 %
46.6 %
27.6 %
72.4 %
14/58
28/58
16/58
42/58
24.1 %
48.3 %
27.6 %
72.4 %
16/58
28/58
14/58
44/58
27.6 %
48.3 %
24.1 %
75.9 %
The ancestral allele 1 of each polymorphism is represented in bold
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This latter condition may be due to abnormalities in the TSHr [5–8] or defective Gsa protein in type 1 pseudohypoparathyroidism [19]. As a result of a genomic-wide study, a strong association between three polymorphic variants in intron 1 of PDE8B gene (rs4704397, rs6885099 and rs2046045) and serum levels of TSH have been recently reported [11]. In particular, each copy of the minor allele A of rs4704397 SNP was found to be associated with an increase of TSH concentration of 0.13 mU/l. A strong association has also been demonstrated for serum TSH levels and SNPs rs6885099 and rs2046045 [11]. A meta-analysis confirmed that genetic variation in PDE8B was associated with TSH and identified a possible new association with free T4 [12]; this study also demonstrated the continuing association between genetic variation due to SNPs on TSH and thyroid hormone levels [12]. The rs4704397 SNP of PDE8B gene has also been demonstrated to be associated with SH in pregnancy [13] and with serum TSH levels in a cohort of obese children [14]. In this paper, we studied the frequency of PDE8B gene polymorphisms in a large series of subjects with sporadic NSH where no TSHr gene mutations were identified. Studying a total of 116 alleles, we identified 84 ancestral alleles associated with increased TSH level for rs4704397 polymorphism (72.4 %), 84 ancestral alleles for rs6885099 polymorphism (72.4 %) and 88 ancestral alleles for rs2046045 polymorphism (75.9 %). Similar values of serum TSH were detected in patients with minor or major alleles for each polymorphism demonstrating a lack of association between the polymorphisms and circulating
Statistical analysis was conducted to identify any difference in the circulating serum TSH values for the three genotypes of each polymorphism. For each studied polymorphism, median values of circulating serum TSH level were similar in patients with at least one copy of the allele associated with elevated TSH and those without this allele (Fig. 1). The further subdivision of patients in a group with serum TSH between 3.6 and 10 lU/ml and a group with serum TSH [ 10 lU/ml did not show statistically significant differences in circulating TSH with respect to the studied PDE8B gene polymorphisms (data not shown).
Discussion Subclinical hypothyroidism is a clinical condition characterized by an elevated serum TSH level associated with normal FT4 and FT3. The patient is usually asymptomatic [1], but careful evaluation has shown consequences on quality of life, cognitive abilities, cholesterol metabolism, heart rate, bone mineral density and atherogenesis [1, 2]. Furthermore, SH progresses toward clinically overt hypothyroidism in a substantial portion of patients [2]. Chronic autoimmune thyroiditis is the most frequent cause of SH in adults in western countries [2] and its diagnostic hallmarks are circulating anti-thyroid antibodies and a hypoechogenic pattern of thyroid gland at ultrasound examination [18]. Autoimmune SH must be distinguished from the rare nonautoimmune condition of thyroid resistance to TSH, a syndrome in which the thyroid shows a variable degree of hyposensitivity to a biological active TSH molecule [3].
rs6885099
rs4704397
40
40
35
35
Serum TSH (mU/l)
Serum TSH (mU/l)
Fig. 1 Boxplots showing the distribution of TSH serum levels related to genotype of rs4704397, rs6885099 and rs2046045 polymorphisms. The upper and the lower limits of the boxes represent the 75th and the 25th percentile, respectively; the lines inside the boxes indicate the median. The upper and lower whiskers indicate 90th and 10th percentile, respectively. Outliers are illustrated as full circles
30 25 20 15 10 5
30 25 20 15 10 5
0
0
AA
AG
GG
GG rs2046045
40
Serum TSH (mU/l)
35 30 25 20 15 10 5 0
GG
123
GT
TT
GA
AA
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TSH levels: in these patients, the presence of the studied polymorphisms did not determine a further increase in TSH levels. It could be assumed, therefore, that in patients affected by idiopathic NSH, where the most frequent cause of this biochemical anomaly was excluded, the presence or absence of rs4704397 polymorphism does not further affect or increase circulating TSH serum level. PDE8B is undetectable in pituitary gland [20] and it acts primarily in the thyroid to catalyze the hydrolysis and inactivation of cAMP after TSH signaling. Variations in PDE8B activity may influence T4 and T3 levels by making the thyroid less responsive to TSH and doing that higher concentrations of TSH are necessary to maintain the homeostasis of thyroid hormones in the blood. It can be inferred that much of the changes in TSH levels affecting normal and possibly pathological states might to be exerted at the level of cAMP degradation [11]. It is possible that genomic PDE8B mutations could be responsible for some cases of NSH with no evidence of thyroid autoimmunity or loss of function mutations in TSHr gene or Gs alpha [11]. Recently, a mutation in PDE8B gene leading to elevated cAMP levels has been identified in a case of adrenal hyperplasia in Cushing syndrome [21] and a complex frameshift mutation of PDE8B gene, resulting in a loss of enzymatic phosphodiesterase activity, has been described as the cause of autosomal-dominant striatal degeneration [22]. Increased cAMP-degradating PDE8B activity has been detected in autonomous thyroid adenomas, where it may represent a compensatory mechanism opposing the constitutive activation of the cAMP pathway [23]. In the light of these findings might be interesting to analyze the entire coding sequence of the PDE8B gene in our patients affected by NSH to possibly identify mutations of the gene responsible for an altered function of the enzyme leading to abnormal levels of cAMP in thyroid and determining an increase of circulating TSH for effect of the negative feedback loop. Recently, another SNP has been shown with convincing significance and rigorous replication to be associated with differences in circulating concentrations of TSH: SNP rs10917469 on chromosome 1p36.13 [24]. This common variant is close to CAPZB gene (MIM *601572) encoding the beta subunit of the barbed-end actin binding protein, a member of the F-actin-capping protein family; it is not known to be involved in thyroid function, but gene expression data (Unigene Hs.432760) reveals high levels of expression in normal thyroid in humans and moderate expression in pituitary, making it biologically plausible that the gene regulates pituitary–thyroid axis function. The difference in mean TSH concentrations between wild-type individuals and those homozygous for the minor G allele was 0.5 mU/l, which is likely to be clinically relevant [24]. In conclusion, a prevalence of minor alleles of PDE8B gene SNPs associated with elevated serum levels of TSH
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was demonstrated in patients affected by NSH. However, a lack of association between the PDE8B gene polymorphisms and TSH levels was demonstrated in patients with idiopathic NSH. Further studies will be conducted to verify the integrity of the PDE8B gene in these patients. Acknowledgments This work was supported by grants from Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica (MURST) and Ministero della Sanita`, Ricerca Finalizzata. We are grateful to MURST and Ministero della Sanita` for funding. Conflict of interest The authors P. Agretti, G. De Marco, C. Di Cosmo, B. Bagattini, E. Ferrarini, L. Montanelli, P. Vitti, and M. Tonacchera declare that they have no conflict of interest.
References 1. Cooper DS (2001) Subclinical hypothyroidism. N Engl J Med 345:260–265 2. Ross DS (2000) Subclinical hypothyroidism. In: Braverman LE, Utiger RD (eds) Werner and ingbar’s the thyroid: a fundamental and clinical text, 8th edn. Lippincott Williams & Wilkins, Philadelphia, pp 467–473 3. Refetoff S (2003) Resistance to thyrotropin. J Endocrinol Invest 26:770–779 4. Stanbury JB, Rocmans P, Buhler UK, Ochi Y (1968) Congenital hypothyroidism with impaired thyroid response to thyrotropin. N Engl J Med 279:1132–1136 5. Sunthornthepvarakui T, Gottschalk ME, Hayashi Y, Refetoff S (1995) Resistance to thyrotropin caused by mutations in the thyrotropin-receptor gene. N Engl J Med 332:155–160 6. Tonacchera M, Agretti P, De Marco G et al (2001) Thyroid resistance to TSH complicated by autoimmune thyroiditis. J Clin Endocrinol Metab 86:4543–4546 7. Tonacchera M, Agretti P, Pinchera A et al (2000) Congenital hypothyroidism with impaired thyroid response to thyrotropin (TSH) and absent circulating thyroglobulin: evidence for a new inactivating mutation of the TSH receptor gene. J Clin Endocrinol Metab 85:1001–1008 8. Persani L, Calebiro D, Cordella D et al (2010) Genetics and phenomics of hypothyroidism due to TSH resistance. Mol Cell Endocrinol 322:72–82 9. Peeters RP, van Toor H, Klootwijk W et al (2003) Polymorphisms in thyroid hormone pathway genes are associated with plasma TSH and iodothyronine levels in healthy subjects. J Clin Endocrinol Metab 88:2880–2888 10. Panicker V, Cluett C, Shields B et al (2008) A common variation in deiodinase 1 gene DIO1 is associated with the relative levels of free thyroxine and triiodothyronine. J Clin Endocrinol Metab 93:3075–3081 11. Arnaud-Lopez L, Usala G, Ceresini G et al (2008) Phosphodiesterase 8B gene variants are associated with serum TSH levels and thyroid function. Am J Hum Genet 82:1270–1280 12. Taylor PN, Panicker V, Sayers A et al (2011) A meta-analysis of the associations between common variation in the PDE8B gene and thyroid hormone parameters, including assessment of longitudinal stability of associations over time and effect of thyroid hormone replacement. Eur J Endocrinol 164:773–780 13. Shields BM, Freathy RM, Knight BA et al (2009) Phosphodiesterase 8B gene polymorphism is associated with subclinical hypothyroidism in pregnancy. J Clin Endocrinol Metab 94: 4608–4612
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194 14. Grandone A, Perrone L, Cirillo G et al (2012) Impact of the phosphodiesterase 8B gene rs4704397 variation on thyroid homeostasis in childhood obesity. Eur J Endocrinol 166:255–260 15. Rotondi M, Leporati P, La Manna A et al (2009) Raised serum TSH levels in patients with morbid obesity: is it enough to diagnose subclinical hypothyroidism? Eur J Endocrinol 160: 403–408 16. Rotondi M, Magri F, Chiovato L (2011) Thyroid and obesity: not a one-way interaction. J Clin Endocrinol Metab 96:344–346 17. Tonacchera M, Banco M, Lapi P et al (2004) Genetic analysis of TTF-2 gene in children with congenital hypothyroidism and cleft palate, congenital hypothyroidism, or isolated cleft palate. Thyroid 14:584–588 18. Marcocci C, Vitti P, Cetani F, Catalano F, Concetti R, Pinchera A (1991) Thyroid ultrasonography helps to identify patients with diffuse lymphocytic thyroiditis who are prone to develop hypothyroidism. J Clin Endocrinol Metab 72:209–213 19. Yokoyama M, Takeda K, Iyota K, Okabayashi T, Hashimoto K (1996) A 4-base pair deletion mutation of Gs alpha gene in a
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20.
21.
22.
23.
24.
Japanese patient with pseudohypoparathyroidism. J Endocrinol Invest 19:236–241 Persani L, Borgato S, Lania A et al (2001) Relevant cAMPspecific phosphodiesterase isoforms in human pituitary: effect of Gs(alpha) mutations. J Clin Endocrinol Metab 86:3795–3800 Horvath A, Mericq V, Stratakis CA (2008) Mutation in PDE8B, a cyclic AMP-specific phosphodiesterase in adrenal hyperplasia. N Engl J Med 358:750–752 Appenzeller S, Schirmacher A, Halfter H et al (2010) Autosomaldominant striatal degeneration is caused by a mutation in the phosphodiesterase 8B gene. Am J Hum Genet 86:83–87 Persani L, Lania A, Alberti L et al (2000) Induction of specific phosphodiesterase isoforms by constitutive activation of the cAMP pathway in autonomous thyroid adenomas. J Clin Endocrinol Metab 85:2872–2878 Panicker V, Wilson SG, Walsh JP et al (2010) A locus on chromosome 1p36 is associated with thyrotropin and thyroid function as identified by genome-wide association study. Am J Hum Genet 87:430–435
ORIGINAL ARTICLE
Frequency and effect on serum TSH of phosphodiesterase 8B (PDE8B) gene polymorphisms in patients with sporadic nonautoimmune subclinical hypothyroidism P. Agretti • G. De Marco • C. Di Cosmo • B. Bagattini • E. Ferrarini • L. Montanelli P. Vitti • M. Tonacchera
•
Received: 10 July 2013 / Accepted: 16 November 2013 / Published online: 9 January 2014 Ó Italian Society of Endocrinology (SIE) 2013
Abstract Background Nonautoimmune subclinical hypothyroidism (NSH) is characterized by elevated serum TSH in presence of normal thyroid hormone levels and absence of antithyroid antibodies. As result of a genomic-wide study, a strong association between three polymorphic variants in intron 1 of human PDE8B gene (rs4704397, rs6885099, rs2046045) and serum TSH has been reported in euthyroid subjects. Aim The aim of this study was to evaluate frequency and effects on serum TSH of PDE8B gene polymorphisms in patients with sporadic NSH and verify if differences in serum TSH levels are associated to these polymorphic variants. Subjects and methods A total of 58 Italian selected patients affected by NSH, with elevated serum TSH, normal FT3 and FT4 and without TSHr gene mutations, were subjected to genotyping for specific single nucleotide polymorphism of PDE8B gene. Results In all patients, the integrity of TSH receptor gene was attested. The ancestral allele associated with increased serum TSH was present in 42/58 patients (72.4 %) for rs4704397, in 42/58 patients (72.4 %) for rs6885099 and in 44/58 patients (75.9 %) for rs2046045. However, similar
P. Agretti G. De Marco C. Di Cosmo B. Bagattini E. Ferrarini L. Montanelli P. Vitti M. Tonacchera Dipartimento di Medicina Clinica e Sperimentale, Sezione di Endocrinologia, Universita` di Pisa, Azienda Ospedaliero Universitaria Pisana, Pisa, Italy M. Tonacchera (&) Dipartimento di Medicina Clinica e Sperimentale, Sezione di Endocrinologia, Universita` di Pisa, Via Paradisa 2, 56124 Pisa, Italy e-mail: [email protected]
values of serum TSH were detected in patients with minor or major allele for each polymorphism. Conclusions A prevalence of the minor allele of PDE8B gene polymorphism associated with elevated serum levels of TSH was demonstrated in patients affected by sporadic NSH; however, significant differences in circulating TSH in patients with minor or major alleles for each polymorphism were not identified demonstrating the lack of association between the polymorphisms and serum TSH levels in these patients. Keywords PDE8B gene Single nucleotide polymorphisms Nonautoimmune subclinical hypothyroidism Circulating TSH
Introduction Subclinical hypothyroidism (SH) defines a condition characterized by elevated serum TSH associated with normal circulating FT4 and FT3 [1]. The most common etiology of SH in western countries is chronic autoimmune thyroiditis that may progress toward clinically overt hypothyroidism [2]. SH due to chronic autoimmune thyroiditis must be distinguished from the rare condition of thyroid resistance to TSH (nonautoimmune subclinical hypothyroidism, NSH), a congenital syndrome of variable hyposensitivity to a biologically active TSH molecule [3]. The defect is characterized by elevated serum TSH and normal to very low serum levels of thyroid hormones in the presence of a hypoplastic or normally sized gland in the proper position in the neck. Depending on the degree of impairment of TSHr function, subjects can present with euthyroid hyperthyrotropinemia at one extreme of the spectrum, or severe hypothyroidism at the other [3, 4].
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Germline loss of function mutations of the TSH receptor gene has been described in the case of partial or complete TSH resistance [5–8]. In the last years, variants of different genes have been associated with susceptibility of normal population to alteration of thyroid function [9, 10]. In particular, three single nucleotide polymorphisms (SNPs) in PDE8B gene (rs4704397, rs6885099, rs2046045) have been found to be strongly associated with altered serum TSH concentrations in general population [11]. By genotyping 362,129 SNPs in 4,300 Sardinians, the authors identified the strongest association between alleles of rs4704397 and circulating serum TSH levels; each additional copy of the minor A allele was associated with an increase of 0.13 lU/ml in TSH. A strong association has also been demonstrated for serum TSH levels and SNPs rs6885099 and rs2046045 [11]. A meta-analysis by Taylor et al. [12] 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. The polymorphism rs4704397 of PDE8B gene was also demonstrated to be associated with SH in pregnancy [13] and with serum TSH levels in obese children [14]. On the other hand obesity, as known, positively correlates with elevated serum concentration of TSH [15, 16]. The PDE8B gene (PDE8B, MIM ?603390) codifies for a thyroid-specific phosphodiesterase, which catalyzes the hydrolysis of cAMP in thyroid and thereby regulates cAMP levels in the gland. From the foregoing, it would affect the production of thyroid hormones and TSH release from pituitary modulating thyroid physiology [11]. PDE8B polymorphisms may reduce cAMP in the thyroid leading to a decreased thyroid stimulatory response to TSH and lower thyroid hormone production. This is likely to result in a higher TSH level required to maintain FT4 and FT3 in the normal range due to the negative feedback [13]. In this paper, we aimed to assess the frequency of the three PDE8B gene polymorphisms showing the strongest association with circulating TSH levels in a group of patients affected by sporadic NSH and to verify if different TSH serum levels were associated with different polymorphic variants of PDE8B in patients with idiopathic NSH.
Materials and methods Study group Our study group consisted of 58 patients affected by NSH. Thirty-five females and 23 males with a mean age of 27.1 ± 14.1 years (ranging from 4 to 73 years, with the same normal range of TSH) were included in the study. In
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all 58 subjects, serum thyroid hormone levels were within the normal range (FT4 mean 10.1 ± 2.1 pg/ml with normal values of 7.0–17.0 pg/ml, FT3 mean 3.9 ± 0.8 pg/ml with normal values of 2.7–5.7 pg/ml) and serum TSH level was elevated (mean TSH 7.6 ± 5.7 lU/ml ranging from 3.6 to 38.0 lU/ml, with a normal range of 0.4–3.4 lU/ml). Serial dilutions of the TSH immunoreactivity were performed in all cases and were always parallel to the standard curve (data not shown). Test for antithyroperoxidase (TPOAb), antithyroglobulin (TgAb) and antiTSH-receptor (TRAb) antibodies gave negative results. Ultrasound of the thyroid showed a normal gland in the proper position in the neck with a normoechogenic pattern. After intravenous TRH challenge, the surge of TSH was proportional to basal TSH values and was followed by an adequate production of FT4 and FT3, suggesting a normal bioactivity of the TSH molecule (data not shown). In all 58 patients, no thyroid defect was identified in the parents and relatives investigated. Subjects of pediatric age received L-T4 therapy, which normalized serum TSH level. Adult patients with NSH were not treated with L-T4 replacement therapy, and serum thyroid hormone and TSH levels were checked once a year. After a follow-up of 3 years, serum TSH concentrations were unchanged with values similar to those recorded at the first observation. None of the subjects developed in vitro or in vivo features of thyroid autoimmunity. Subjects with signs of atherosclerotic cardiovascular disease or with other chronic diseases that could affect endothelial function such as obesity and diabetes were not included in the study group. All 58 patients were comparable with respect to lipid profile and their resting electrocardiograms were normal. In Table 1, the anthropometric and clinical characteristics of the patients are represented. The study was approved by the local ethical committee, and informed consent was obtained from all subjects. Laboratory evaluation of thyroid function Serum FT3 and FT4 were measured with a chemiluminescent method (Vitro System, Ortho-Clinical Diagnostics, Rochester, NY, USA). Thyrotropin (TSH) was assessed by Table 1 Anthropometric and clinical features of patients affected by sporadic or familial nonautoimmune subclinical hypothyroidism Age (years)
27.1 ± 14.1
Weight (kg)
58.9 ± 15.1
Height (cm)
157.3 ± 22.3
BMI TSH (lU/ml)
23.4 ± 2.1 7.6 ± 5.7
FT3 (pg/ml)
3.9 ± 0.8
FT4 (pg/ml)
10.1 ± 2.1
Average values and standard deviations are reported
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ultrasensitive commercial chemiluminescent method (Immulite 2000; Diagnostic Products, Los Angeles, CA, USA). TPOAb and TgAb were measured using a two-step immunoenzymatic assay (AIA-Pack TgAb and TPOAb; Tosoh, Tokyo, Japan). Serum Tg was measured using an immunometric chemiluminescence assay (Immulite 2000; Diagnostic Products, Los Angeles, CA, USA). TSH receptor (TSHr) antibodies were measured using a commercial radioreceptor assay (TRAK assay, B.R.A.H.M.S., Berlin, Germany).
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population, and in our case permitted to assess differences between the three genotypes of polymorphisms rs4704397, rs6885099 and rs2046045. The critical value for the Kruskal–Wallis H test comes from the Chi-square table with k - 1 degrees of freedom, where k is the number of groups being tested. The computed value must be larger than the critical value in order to reject the null hypothesis.
Results Sequence determination and genotyping Genomic DNA was extracted from peripheral lymphocytes of the patients using standard procedures [17]. All coding exons of the TSHr gene were PCR amplified and sequenced using previously described methods [6], and the sequencing products were analyzed on a 3130xl Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Fifty nanograms of genomic DNA were used to genotype patients for specific SNPs of PDE8B gene by predeveloped TaqMan SNP genotyping assay (Applied Biosystems, Foster City, CA, USA). Allele-specific probes were labeled with the fluorescent dyes VIC and FAM, respectively. PCR was carried out in a total reaction volume of 20 ll with the following amplification protocol: denaturation at 95 °C for 10 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 1 min. To obtain a high confidence level, the manufacturer recommended to run for each assay four no-template controls, four allele 1 controls and four allele 2 controls. The ABI PRIM 7700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) was used to detect and measure the fluorescence signals generated from the PCR amplification during an endpoint plate read. After the plate read was completed, SDS software calculated the fluorescence measurements made during the plate read and plotted fluorescence values based on signals from each well; in this way, the genotype of each sample and control was determined. A positive signal indicates a perfect match between the probe and the tested DNA, thus identifying the allele types. Statistical analysis Circulating serum levels of TSH for the study group were not normally distributed, while FT3 and FT4 values were approximately normally distributed. Descriptive statistics for the study group are presented as median and interquartile range, and nonparametric analysis was carried out using the Kruskall–Wallis H test [13]. This method permits to assess differences between median values of different groups where there is not a normal distribution of
We selected 58 patients with sporadic NSH who did not harbor TSHr mutations. In five patients, the polymorphic variant P52T of the TSHr was present, while in one patient the D36H and P52T polymorphic variants of the TSHr were simultaneously present. All genetic variants of TSHr gene were identified at the heterozygous state (data not shown). The haplotype associated with increased serum TSH level is A for rs4704397, G for rs6885099 and G for rs2046045 and each one corresponds to the ancestral allele. The ancestral allele A of rs4704397, the polymorphism of PDE8B gene which shows the strongest association to elevated circulating levels of serum TSH, was present in 42/58 subjects corresponding to about 72 %; this allele was present in the homozygous state (AA) in 15/58 subjects (25.8 %) and in the heterozygous state (AG) in 27/58 subjects (46.6 %). The ancestral allele G of rs6885099 PDE8B gene polymorphism was present in 42/58 subjects corresponding to about 72 %; this allele was present in the homozygous state (GG) in 14/58 subjects (24.1 %) and in the heterozygous state (GA) in 28/58 subjects (48.3 %). The ancestral allele G of rs2046045 PDE8B gene polymorphism was present in 44/58 subjects corresponding to about 76 %; this allele was present in the homozygous state (GG) in 16/58 subjects (27.6 %) and in the heterozygous state (GT) in 28/58 subjects (48.3 %) (Table 2).
Table 2 Frequency of PDE8B polymorphisms in patients affected by sporadic or familial nonautoimmune subclinical hypothyroidism
rs4704397 (A/G) rs6885099 (G/A) rs2046045 (G/T)
Allele 1
Allele 1/2
Allele 2
Total allele 1
15/58
27/58
16/58
42/58
25.8 %
46.6 %
27.6 %
72.4 %
14/58
28/58
16/58
42/58
24.1 %
48.3 %
27.6 %
72.4 %
16/58
28/58
14/58
44/58
27.6 %
48.3 %
24.1 %
75.9 %
The ancestral allele 1 of each polymorphism is represented in bold
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This latter condition may be due to abnormalities in the TSHr [5–8] or defective Gsa protein in type 1 pseudohypoparathyroidism [19]. As a result of a genomic-wide study, a strong association between three polymorphic variants in intron 1 of PDE8B gene (rs4704397, rs6885099 and rs2046045) and serum levels of TSH have been recently reported [11]. In particular, each copy of the minor allele A of rs4704397 SNP was found to be associated with an increase of TSH concentration of 0.13 mU/l. A strong association has also been demonstrated for serum TSH levels and SNPs rs6885099 and rs2046045 [11]. A meta-analysis confirmed that genetic variation in PDE8B was associated with TSH and identified a possible new association with free T4 [12]; this study also demonstrated the continuing association between genetic variation due to SNPs on TSH and thyroid hormone levels [12]. The rs4704397 SNP of PDE8B gene has also been demonstrated to be associated with SH in pregnancy [13] and with serum TSH levels in a cohort of obese children [14]. In this paper, we studied the frequency of PDE8B gene polymorphisms in a large series of subjects with sporadic NSH where no TSHr gene mutations were identified. Studying a total of 116 alleles, we identified 84 ancestral alleles associated with increased TSH level for rs4704397 polymorphism (72.4 %), 84 ancestral alleles for rs6885099 polymorphism (72.4 %) and 88 ancestral alleles for rs2046045 polymorphism (75.9 %). Similar values of serum TSH were detected in patients with minor or major alleles for each polymorphism demonstrating a lack of association between the polymorphisms and circulating
Statistical analysis was conducted to identify any difference in the circulating serum TSH values for the three genotypes of each polymorphism. For each studied polymorphism, median values of circulating serum TSH level were similar in patients with at least one copy of the allele associated with elevated TSH and those without this allele (Fig. 1). The further subdivision of patients in a group with serum TSH between 3.6 and 10 lU/ml and a group with serum TSH [ 10 lU/ml did not show statistically significant differences in circulating TSH with respect to the studied PDE8B gene polymorphisms (data not shown).
Discussion Subclinical hypothyroidism is a clinical condition characterized by an elevated serum TSH level associated with normal FT4 and FT3. The patient is usually asymptomatic [1], but careful evaluation has shown consequences on quality of life, cognitive abilities, cholesterol metabolism, heart rate, bone mineral density and atherogenesis [1, 2]. Furthermore, SH progresses toward clinically overt hypothyroidism in a substantial portion of patients [2]. Chronic autoimmune thyroiditis is the most frequent cause of SH in adults in western countries [2] and its diagnostic hallmarks are circulating anti-thyroid antibodies and a hypoechogenic pattern of thyroid gland at ultrasound examination [18]. Autoimmune SH must be distinguished from the rare nonautoimmune condition of thyroid resistance to TSH, a syndrome in which the thyroid shows a variable degree of hyposensitivity to a biological active TSH molecule [3].
rs6885099
rs4704397
40
40
35
35
Serum TSH (mU/l)
Serum TSH (mU/l)
Fig. 1 Boxplots showing the distribution of TSH serum levels related to genotype of rs4704397, rs6885099 and rs2046045 polymorphisms. The upper and the lower limits of the boxes represent the 75th and the 25th percentile, respectively; the lines inside the boxes indicate the median. The upper and lower whiskers indicate 90th and 10th percentile, respectively. Outliers are illustrated as full circles
30 25 20 15 10 5
30 25 20 15 10 5
0
0
AA
AG
GG
GG rs2046045
40
Serum TSH (mU/l)
35 30 25 20 15 10 5 0
GG
123
GT
TT
GA
AA
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TSH levels: in these patients, the presence of the studied polymorphisms did not determine a further increase in TSH levels. It could be assumed, therefore, that in patients affected by idiopathic NSH, where the most frequent cause of this biochemical anomaly was excluded, the presence or absence of rs4704397 polymorphism does not further affect or increase circulating TSH serum level. PDE8B is undetectable in pituitary gland [20] and it acts primarily in the thyroid to catalyze the hydrolysis and inactivation of cAMP after TSH signaling. Variations in PDE8B activity may influence T4 and T3 levels by making the thyroid less responsive to TSH and doing that higher concentrations of TSH are necessary to maintain the homeostasis of thyroid hormones in the blood. It can be inferred that much of the changes in TSH levels affecting normal and possibly pathological states might to be exerted at the level of cAMP degradation [11]. It is possible that genomic PDE8B mutations could be responsible for some cases of NSH with no evidence of thyroid autoimmunity or loss of function mutations in TSHr gene or Gs alpha [11]. Recently, a mutation in PDE8B gene leading to elevated cAMP levels has been identified in a case of adrenal hyperplasia in Cushing syndrome [21] and a complex frameshift mutation of PDE8B gene, resulting in a loss of enzymatic phosphodiesterase activity, has been described as the cause of autosomal-dominant striatal degeneration [22]. Increased cAMP-degradating PDE8B activity has been detected in autonomous thyroid adenomas, where it may represent a compensatory mechanism opposing the constitutive activation of the cAMP pathway [23]. In the light of these findings might be interesting to analyze the entire coding sequence of the PDE8B gene in our patients affected by NSH to possibly identify mutations of the gene responsible for an altered function of the enzyme leading to abnormal levels of cAMP in thyroid and determining an increase of circulating TSH for effect of the negative feedback loop. Recently, another SNP has been shown with convincing significance and rigorous replication to be associated with differences in circulating concentrations of TSH: SNP rs10917469 on chromosome 1p36.13 [24]. This common variant is close to CAPZB gene (MIM *601572) encoding the beta subunit of the barbed-end actin binding protein, a member of the F-actin-capping protein family; it is not known to be involved in thyroid function, but gene expression data (Unigene Hs.432760) reveals high levels of expression in normal thyroid in humans and moderate expression in pituitary, making it biologically plausible that the gene regulates pituitary–thyroid axis function. The difference in mean TSH concentrations between wild-type individuals and those homozygous for the minor G allele was 0.5 mU/l, which is likely to be clinically relevant [24]. In conclusion, a prevalence of minor alleles of PDE8B gene SNPs associated with elevated serum levels of TSH
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was demonstrated in patients affected by NSH. However, a lack of association between the PDE8B gene polymorphisms and TSH levels was demonstrated in patients with idiopathic NSH. Further studies will be conducted to verify the integrity of the PDE8B gene in these patients. Acknowledgments This work was supported by grants from Ministero dell’Universita` e della Ricerca Scientifica e Tecnologica (MURST) and Ministero della Sanita`, Ricerca Finalizzata. We are grateful to MURST and Ministero della Sanita` for funding. Conflict of interest The authors P. Agretti, G. De Marco, C. Di Cosmo, B. Bagattini, E. Ferrarini, L. Montanelli, P. Vitti, and M. Tonacchera declare that they have no conflict of interest.
References 1. Cooper DS (2001) Subclinical hypothyroidism. N Engl J Med 345:260–265 2. Ross DS (2000) Subclinical hypothyroidism. In: Braverman LE, Utiger RD (eds) Werner and ingbar’s the thyroid: a fundamental and clinical text, 8th edn. Lippincott Williams & Wilkins, Philadelphia, pp 467–473 3. Refetoff S (2003) Resistance to thyrotropin. J Endocrinol Invest 26:770–779 4. Stanbury JB, Rocmans P, Buhler UK, Ochi Y (1968) Congenital hypothyroidism with impaired thyroid response to thyrotropin. N Engl J Med 279:1132–1136 5. Sunthornthepvarakui T, Gottschalk ME, Hayashi Y, Refetoff S (1995) Resistance to thyrotropin caused by mutations in the thyrotropin-receptor gene. N Engl J Med 332:155–160 6. Tonacchera M, Agretti P, De Marco G et al (2001) Thyroid resistance to TSH complicated by autoimmune thyroiditis. J Clin Endocrinol Metab 86:4543–4546 7. Tonacchera M, Agretti P, Pinchera A et al (2000) Congenital hypothyroidism with impaired thyroid response to thyrotropin (TSH) and absent circulating thyroglobulin: evidence for a new inactivating mutation of the TSH receptor gene. J Clin Endocrinol Metab 85:1001–1008 8. Persani L, Calebiro D, Cordella D et al (2010) Genetics and phenomics of hypothyroidism due to TSH resistance. Mol Cell Endocrinol 322:72–82 9. Peeters RP, van Toor H, Klootwijk W et al (2003) Polymorphisms in thyroid hormone pathway genes are associated with plasma TSH and iodothyronine levels in healthy subjects. J Clin Endocrinol Metab 88:2880–2888 10. Panicker V, Cluett C, Shields B et al (2008) A common variation in deiodinase 1 gene DIO1 is associated with the relative levels of free thyroxine and triiodothyronine. J Clin Endocrinol Metab 93:3075–3081 11. Arnaud-Lopez L, Usala G, Ceresini G et al (2008) Phosphodiesterase 8B gene variants are associated with serum TSH levels and thyroid function. Am J Hum Genet 82:1270–1280 12. Taylor PN, Panicker V, Sayers A et al (2011) A meta-analysis of the associations between common variation in the PDE8B gene and thyroid hormone parameters, including assessment of longitudinal stability of associations over time and effect of thyroid hormone replacement. Eur J Endocrinol 164:773–780 13. Shields BM, Freathy RM, Knight BA et al (2009) Phosphodiesterase 8B gene polymorphism is associated with subclinical hypothyroidism in pregnancy. J Clin Endocrinol Metab 94: 4608–4612
123
194 14. Grandone A, Perrone L, Cirillo G et al (2012) Impact of the phosphodiesterase 8B gene rs4704397 variation on thyroid homeostasis in childhood obesity. Eur J Endocrinol 166:255–260 15. Rotondi M, Leporati P, La Manna A et al (2009) Raised serum TSH levels in patients with morbid obesity: is it enough to diagnose subclinical hypothyroidism? Eur J Endocrinol 160: 403–408 16. Rotondi M, Magri F, Chiovato L (2011) Thyroid and obesity: not a one-way interaction. J Clin Endocrinol Metab 96:344–346 17. Tonacchera M, Banco M, Lapi P et al (2004) Genetic analysis of TTF-2 gene in children with congenital hypothyroidism and cleft palate, congenital hypothyroidism, or isolated cleft palate. Thyroid 14:584–588 18. Marcocci C, Vitti P, Cetani F, Catalano F, Concetti R, Pinchera A (1991) Thyroid ultrasonography helps to identify patients with diffuse lymphocytic thyroiditis who are prone to develop hypothyroidism. J Clin Endocrinol Metab 72:209–213 19. Yokoyama M, Takeda K, Iyota K, Okabayashi T, Hashimoto K (1996) A 4-base pair deletion mutation of Gs alpha gene in a
123
J Endocrinol Invest (2014) 37:189–194
20.
21.
22.
23.
24.
Japanese patient with pseudohypoparathyroidism. J Endocrinol Invest 19:236–241 Persani L, Borgato S, Lania A et al (2001) Relevant cAMPspecific phosphodiesterase isoforms in human pituitary: effect of Gs(alpha) mutations. J Clin Endocrinol Metab 86:3795–3800 Horvath A, Mericq V, Stratakis CA (2008) Mutation in PDE8B, a cyclic AMP-specific phosphodiesterase in adrenal hyperplasia. N Engl J Med 358:750–752 Appenzeller S, Schirmacher A, Halfter H et al (2010) Autosomaldominant striatal degeneration is caused by a mutation in the phosphodiesterase 8B gene. Am J Hum Genet 86:83–87 Persani L, Lania A, Alberti L et al (2000) Induction of specific phosphodiesterase isoforms by constitutive activation of the cAMP pathway in autonomous thyroid adenomas. J Clin Endocrinol Metab 85:2872–2878 Panicker V, Wilson SG, Walsh JP et al (2010) A locus on chromosome 1p36 is associated with thyrotropin and thyroid function as identified by genome-wide association study. Am J Hum Genet 87:430–435
