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-Subunit Gs Is Imprinted in Human Thyroid Glands: Implications for Thyroid Function in Pseudohypoparathyroidism Types 1A and 1B
Metabolic Diseases Branch, National Institute of Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892
Address all correspondence and requests for reprints to: Dr. Lee S. Weinstein, Metabolic Diseases Branch, National Institute of Diabetes, Digestive, and Kidney Diseases, National Institutes of Health, Building 10, Room 8C101, Bethesda, Maryland 20892-1752. E-mail: leew{at}amb.niddk.nih.gov.
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Abstract |
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-subunit Gs couples receptors to adenylyl cyclase and is required for hormone-stimulated cAMP generation. In Albright hereditary osteodystrophy, heterozygous Gs null mutations only lead to PTH, TSH, and gonadotropin resistance when inherited maternally [pseudohypoparathyroidism type 1A; (PHP1A)]. Maternal-specific expression of Gs in specific hormone targets could explain this observation. Using hot-stop PCR analysis on total RNA from six normal human thyroid specimens, we showed that the majority of the Gs mRNA (72 ± 3%) was derived from the maternal allele. This is consistent with the presence of TSH resistance in patients with maternal Gs null mutations (PHP1A) and the absence of TSH resistance in patients with paternal Gs mutations (pseudopseudohypoparathyroidism). Patients with PTH resistance in the absence of Albright hereditary osteodystrophy (PHP1B) have an imprinting defect of the Gs gene resulting in both alleles having a paternal epigenotype, which would lead to a more moderate level of thyroid-specific Gs deficiency. We found evidence of borderline TSH resistance in 10 of 22 PHP1B patients. This study provides further evidence for tissue-specific imprinting of Gs in humans and provides a potential mechanism for mild to moderate TSH resistance in PHP1A and borderline resistance in some patients with PHP1B. |
Introduction |
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-, ß-, and 
-subunit, each is the product of separate genes, and is defined by its -subunit, which binds guanine nucleotide and interacts with both receptors and effectors (1). The stimulatory G protein -subunit Gs is ubiquitously expressed and couples heptahelical receptors for hormones and other extracellular signals to various intracellular effectors, including the enzyme adenylyl cyclase. Gs is therefore required for the intracellular cAMP response to various peptide and glycoprotein hormones. Activating Gs mutations result in McCune-Albright syndrome, fibrous dysplasia, or endocrine tumors, whereas heterozygous null mutations lead to Albright hereditary osteodystrophy (AHO) or progressive osseous heteroplasia (1).
AHO is characterized by a constellation of abnormal physical features, including short stature, brachydactyly, osteoma cutis, obesity, rounded facies, and, in some cases, mental or developmental abnormalities (2, 3). Patients who inherit Gs null mutations from their father develop AHO alone, a condition also referred to as pseudopseudohypoparathyroidism (PPHP). In contrast, patients who inherit the same mutations from their mother develop AHO plus multihormone resistance, a condition also referred to as pseudohypoparathyroidism type 1A (PHP1A) (1, 4). The multihormone resistance in PHP1A primarily involves PTH resistance in the kidney (5, 6), TSH resistance in the thyroid (7, 8, 9), and partial gonadotropin resistance in the gonads (10). All of these hormones activate Gs and cAMP generation in their respective target tissues. PHP1A patients present with elevated TSH levels at birth (11, 12, 13) and eventually develop mild TSH resistance, manifested by mildly to moderately elevated circulating TSH levels (typically in the range of 5–20 µIU/ml) with low or low normal thyroid hormone levels and no goiter. The TSH response to TRH is greater than normal despite the fact that the T3 response is below normal (7, 8). A defect in TSH-stimulated adenylyl cyclase activation was confirmed in thyroid membranes obtained from one PHP1A patient (14).
Based on the presence of multihormone resistance in patients with maternal, but not paternal, inheritance of AHO, it was proposed that Gs is imprinted (4). Imprinting is an epigenetic phenomenon affecting a small number of genes that leads to differences in expression between the two parental alleles and is usually associated with differential DNA methylation of the parental alleles within the affected gene (15). If Gs is primarily expressed from the maternal allele in hormone target tissues, then mutations on the active maternal allele would lead to significant loss of Gs expression and hormone resistance, whereas mutations on the inactive paternal allele would have relatively little effect on Gs expression or hormone signaling. This is consistent with the fact that PTH-stimulated urinary cAMP responses are markedly reduced in PHPIA, but are unaffected in PPHP (6). Gs imprinting would have to be tissue specific, because Gs has been shown to be biallelically expressed in several human tissues (16, 17, 18). This explains why in many tissues Gs expression is similarly reduced by about 50% in both PHPIA and PPHP patients (6, 19). Studies in Gs knockout mice confirm that Gs is imprinted in a tissue-specific manner, being imprinted in renal proximal tubules (the major site of PTH action in the kidney), but not in most other tissues (20). Although disruption of the maternal allele in mice leads to mild PTH resistance, TSH and thyroid hormone levels in these mice were normal (21). Recent studies have confirmed the tissue-specific imprinting of Gs in humans (22, 23, 24).
The Gs gene GNAS (formerly GNAS1) at 20q13 consists of 13 coding exons for Gs, as well as 3 additional upstream promoters that generate alternative gene products which themselves are imprinted. The NESP55 and XLs promoters (located 35 and 47 kb upstream of the Gs promoter, respectively) are oppositely imprinted (16, 17, 25). NESP55 is expressed from the maternal allele, whereas XLs is only expressed from the paternal allele. Exon 1A is another alternative first exon located 2.5 kb upstream of Gs exon 1 that generates transcripts that are presumed to be untranslated. Exon 1A transcripts are only generated from the paternal allele, as exon 1A and its promoter are methylated on the maternal allele (26, 27). The Gs promoter itself is unmethylated (17, 27). Patients with PHP1B, who have renal PTH resistance in the absence of AHO, have a GNAS imprinting defect that results in a paternal-specific imprinting pattern in the differentially methylated exon 1A region (both alleles being unmethylated and transcriptionally active) (26, 28). The presence of two alleles with a paternal-specific epigenotype presumably leads to reduced Gs expression in renal proximal tubules and PTH resistance. However, it has been generally assumed that TSH resistance is not a feature typically present in PHP1B patients (7).
Based on the fact that TSH resistance occurs in PHPIA, but not in PPHP (6, 29, 30, 31, 32, 33, 34, 35, 36, 37), we predicted that Gs is imprinted in human thyroid glands. To test this hypothesis, we determined the relative amount of Gs mRNA derived from the maternal and paternal alleles from human thyroid samples that were heterozygous for a silent Fok1 polymorphism within exon 5 of the Gs coding region. Our results demonstrate that Gs is preferentially expressed from the maternal allele in human thyroid glands. The presence of two GNAS alleles with a paternal epigenotype in PHPIB patients should lead to a more mild form of thyroid-specific Gs deficiency. Consistent with this, evidence of mild TSH resistance was present in 10 of 22 PHP1B patients who were confirmed to have the GNAS imprinting defect.
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Patients and Methods |
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To examine Gs imprinting in human thyroid, small thyroid biopsies were performed during neck exploration in patients undergoing parathyroidectomy. All patients had normal TSH and free T4, normal thyroid ultrasound, and no prior thyroid resection.
All patients diagnosed with PHP1B presented with evidence of PTH resistance (hypocalcemia, hyperphosphatemia, elevated PTH levels, and normal serum 25-hydroxyvitamin D levels) without features of AHO. The diagnosis was confirmed by Southern analysis of genomic DNA isolated from blood samples using methylation-sensitive restriction enzymes (26), which demonstrated the loss of maternal-specific methylation of the GNAS exon 1A promoter region in all patients. All studies were performed after informed consent was obtained, and all research protocols were approved by the NIDDK/NIAMS institutional review board.
Analysis of allele-specific expression of Gs by hot-stop PCR
The relative abundance of maternal- and paternal-specific Gs transcripts was determined by hot-stop PCR (38). Total blood RNA was isolated using the QIAamp RNA Blood Mini Kit (Qiagen, Valencia, CA), and total thyroid RNA was isolated by the TRIzol method (Invitrogen, Carlsbad, CA). Total RNA (1 µg) was amplified by RT-PCR as previously described (27) using either a Gs exon 1-specific (5'-CCATGGGCTGCCTCGGGAACA-3') or exon 1A-specific (5'-GGACACTCAGTCGCGTCGGCA-3') upstream primer and a common downstream primer complementary to exon 6 (5'-CCTTTGCATGCTCATAGAATTC-3'). The PCR cycling profile consisted of an initial 5-min denaturation at 95 C, followed by 35 cycles of annealing (60 C, 30 sec), extension (72 C, 60 sec), and denaturation (95 C, 30 sec). The downstream primer was 5' end-labeled with T4 polynucleotide kinase (Fermentas, Hanover, MD) and purified using Microspin columns (Amersham Pharmacia Biotech, Piscataway, NJ). After the final PCR cycle, 1 µl labeled downstream primer (
1600 total cpm) was added to each reaction, followed by one cycle of denaturation (95 C, 5 min), annealing (60 C, 45 sec), and extension (72 C, 10 min). PCR products (10 µl of 100 µl total reaction) were digested with EcoN1 (New England Biolabs, Beverly, MA) in a total volume of 100 µl for 2 h at 37 C. Half of this digest was further digested with Fok1 for 1 h at 37 C. All samples (both single- and double-digested) underwent heat inactivation at 65 C, followed by phenol-chloroform extraction. Samples were separated on a nondenaturing 8% acrylamide gel and exposed to x-ray film. Bands were quantified using a BAS1000 phosphorimager (FujiSystems, Stamford, CT).
Northern analysis
Total RNA isolated from three human thyroid specimens (10 µg/lane) were separated on 1.2% agarose/6.66% formaldehyde gels and transferred onto nylon membranes (Nytran Super Charge, Schleicher & Schuell, Keene, NH). Human Gs and XLs-specific cDNA probes were generated by RT-PCR using the following sets of upstream and downstream primers: 5'-TGCTGGAGAATCTGGTAAAAG-3' (Gs exon 2) and 5'-CCTTGGCATGCTCATAGAATTC-3' (Gs exon 6) for Gs; and 5'-GGATGCCTCCGCTGGTTTCAG-3' and 5'-GTCCAGCTTACGGTTGCTGG-3' for XLs exon 1. Probes were labeled with 32P by random priming using the Radprime DNA labeling system (Invitrogen). The membrane was incubated with the Gs probe in QuickHyb hybridization solution (Stratagene, La Jolla, CA) at 68 C for 2 h and then washed twice with 2x SSC (1x SSC is 0.15 M NaCl and 0.015 M sodium citrate)/0.1% sodium dodecyl sulfate at room temperature for 30 min and once with 0.1x SSC/0.1% sodium dodecyl sulfate at 60 C for 1 h. After exposure to x-ray film and phosphorimager, membranes were stripped by boiling in 0.1x SSC/0.1% sodium dodecyl sulfate for 30 min and rehybridized with the XLs probe in Hybrisol II solution (Intergen, Purchase, NY) at 68 C overnight. Membranes were washed using the same conditions as those used for the Gs probe.
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Results |
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is predominantly expressed from the maternal allele in normal human thyroid
Histologically normal thyroid specimens obtained during neck surgery were immediately frozen in liquid nitrogen. A small portion of each sample was genotyped for the common Fok1 polymorphism located within the coding region of exon 5 (39), and six samples that were heterozygous for the polymorphism (Fok1+/-) were used for Gs expression studies. Total RNA was isolated from each sample and then reversed transcribed using a poly(deoxythymidine) primer. Exon 1A- or Gs-specific partial cDNA products were PCR-amplified from the RT products in separate reactions using exon 1A or Gs exon 1-specific upstream primers, respectively, with a common downstream primer complementary to Gs exon 6 (Fig. 1
). Electrophoresis and ethidium bromide staining of the RT-PCR products showed multiple bands, as would be expected due to alternative splicing of Gs exon 3 (40, 41) (data not shown). No bands were detected in parallel reactions in which reverse transcriptase was not added, ruling out DNA contamination. 32P-Labeled downstream primer was added to the PCR tubes before final denaturation, annealing, and extension to generate homoduplexes that were radioactively labeled at the 3' end.
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exon 4, digestion of all samples with EcoN1 alone produced the expected single 156-bp labeled band. Because exon 1A-specific transcripts are only expressed from the paternal allele, parental origin of the Fok1+ and Fok1- alleles can be assigned by double digestion of the exon 1A-specific RT-PCR products with EcoN1 and Fok1. In thyroid samples A and B, Fok1 fully digests the EcoN1 band to the expected 78-bp band, indicating that the paternal allele is Fok1+ in these samples. In contrast, the exon 1A-specific products generated from the blood RNA sample and thyroid samples C, D, E, and F are not digested by Fok1, indicating that the paternal allele is Fok1- in these samples (the exon 1A-specific products for thyroid sample F were not run on the gel shown in Fig. 1
). For each sample the proportion of Gs transcripts derived from the Fok1+ and Fok1- alleles can be determined by the relative intensities of the two bands after EcoN1 and Fok1 digestion. In the blood sample the two bands are of similar intensity as expected, because Gs is not imprinted in blood cells. In all thyroid samples the intensity of the band derived from the maternal allele was significantly greater than that derived from the paternal allele regardless of whether the maternal allele was Fok1+ or Fok1-. Quantification of the bands revealed that, on average 72 ± 3% of the Gs mRNA transcripts were generated from the maternal allele [P < 0.05 vs. expected (50%); range, 62–77%]. Direct sequencing of the RT-PCR products showed results consistent with those shown in Fig. 1 (data not shown). These findings confirm that Gs is imprinted in human thyroids with the majority of Gs generated from the maternal allele and are consistent with previously published reports (23, 24).
XLs is expressed at very low levels in human thyroid
The paternally expressed Gs isoform XLs has been shown to be capable of mediating a TSH-stimulated cAMP response when cotransfected with the TSH receptor in a nonthyroid cell (42). We therefore examined the relative expression of Gs and XLs in human thyroids to determine whether XLs is likely to significantly contribute to TSH signaling in thyroid. We performed Northern analysis on total RNA from three human thyroid specimens and initially hybridized with a cDNA probe spanning Gs exons 2–6 (Fig. 2). Because these exons are common for both Gs and XLs mRNA, the probe should hybridize to both mRNAs in proportion to their relative abundance. We observed a very prominent 1.8-kb band that represents Gs mRNA as well as two very faint upper bands. Hybridization with a cDNA probe that only spanned the XLs-specific first exon demonstrated a very faint band that coincided with the lower of the two faint upper bands seen with the Gs exon 2–6 probe. By determining the relative intensity of the Gs and XLs-specific bands using a phosphorimager, we estimate that steady state levels of XLs mRNA are only 2–3% of the levels of Gs mRNA.
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We originally reported 13 PHP1B patients with renal PTH resistance in the absence of AHO who had GNAS imprinting with loss of exon 1A imprinting (26) and have subsequently confirmed the same imprinting defect in 9 additional PHP1B patients. This imprinting defect results in both alleles having a paternal-specific genotype, with no methylation of the exon 1A promoter region and biallelic expression of exon 1A-specific transcripts (Fig. 3). Because Gs expression is normally expressed poorly from the paternal allele, one might predict that PHP1B patients should have partial loss of Gs expression in the thyroid, which may lead to mild TSH resistance (Fig. 3). Within our cohort of 22 PHP1B patients with the exon 1A methylation defect, 10 patients have evidence of borderline or mild TSH resistance based on the presence of mildly elevated TSH levels on at least 1 occasion with low or low normal free T4 levels (Table 1). One patient (patient 6) was previously diagnosed with hypothyroidism by an outside physician and was begun on levothyroxine before our evaluation. Another patient (no. 9) developed antithyroid antibodies and thyroid cancer years after his initial diagnosis of hypothyroidism. Otherwise goiter was absent in all patients, and antithyroid antibodies were absent in all other patients (except in patient 4, in whom they were not measured). Although a minority of PHP1B patients also have abnormal imprinting of the NESP55 and XLs upstream regions within the GNAS locus (26), there was no obvious correlation between the presence of TSH abnormalities and the imprinting status of these 2 upstream regions within our cohort (data not shown).
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Discussion |
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is imprinted in a tissue-specific manner in humans (1, 4), and this has been confirmed in mice (20, 21). The observation that PHP1A patients, who inherit Gs null mutations maternally, develop TSH resistance, whereas PPHP patients, who inherit the same mutations paternally, do not develop TSH resistance (6, 29, 30, 31, 32, 33, 34, 35, 36, 37) could be explained by Gs imprinting in the thyroid with partial suppression of the paternal allele. We directly examined allele-specific expression of Gs in normal human thyroid specimens and showed that in all cases Gs mRNA is predominantly, although not exclusively, derived from the maternal allele. It is possible that the extent of Gs imprinting in thyroid follicular cells was underestimated in our studies because the specimens also contained stromal and vascular cells, where Gs is probably not imprinted. This study is consistent with recently published studies showing partial imprinting of Gs in human thyroid and adds to the growing evidence that Gs is imprinted in a tissue-specific manner in humans (22, 23, 24). Although Gs imprinting has been confirmed in human pituitary, thyroid, and gonad, it has not as yet been confirmed in human renal proximal tubules, the presumed site of the PTH signaling defect in PHP (43, 44). The hot-stop PCR method will be useful to directly examine Gs imprinting in proximal tubules and other human tissues of interest.
PHP1A patients have Gs null mutations on the maternal allele. Because only about 28% of the Gs gene product, on the average, is generated from the paternal allele, these patients would be expected to have significantly reduced Gs-specific signaling by TSH, which is consistent with the presence of TSH resistance in the vast majority of PHP1A patients (7, 9). In contrast, PPHP patients, who have the same mutations on the paternal allele, would be expected to maintain significantly higher Gs levels in thyroid cells (72% of normal; see Fig. 3). Despite the small decrease in thyroid-specific Gs expression in these patients, TSH-Gs signaling is maintained at a level sufficient to prevent the development of clinical TSH resistance (6, 29, 30, 31, 32, 33, 34, 35, 36, 37). Although paternal null mutations also disrupt expression of the Gs isoform XLs, which is also capable of mediating TSH-stimulated cAMP production (42), our results indicate that the relative expression of XLs in thyroid is about 1/50th that of Gs, and therefore, XLs probably contributes little to TSH signaling in vivo.
PHP1B patients have an imprinting defect involving the exon 1A region located just upstream of the Gs promoter. Normally this region is methylated on the maternal allele and unmethylated on the paternal allele (26, 27). We have hypothesized that the exon 1A region has a negative regulatory element (e.g. silencer or insulator) that is sensitive to methylation and therefore suppresses Gs expression only from the paternal allele in a tissue-specific manner (1, 26, 27). In PHP1B the exon 1A region is unmethylated on both parental alleles, and therefore both alleles have a paternal epigenotype (26, 28). These patients should therefore have thyroid-specific Gs expression levels that reflect the presence of two paternal alleles, which typically would generate levels that are approximately 56% of normal. Thus, PHP1B patients would be expected to have Gs levels intermediate between those in PHP1A and PPHP patients.
Our findings would predict that PHP1B patients would have TSH resistance that is generally less severe than in PHP1A. In our cohort, 10 of 22 PHP1B patients with a confirmed exon 1A methylation defect had evidence of TSH resistance, with TSH levels above normal but generally less than 10 µIU/ml in the absence of goiter or antithyroid antibodies. In some cases TSH levels were only elevated intermittently, suggesting that subtle resistance can be missed if the levels are only measured on a single occasion. Although one study concluded that PHP1B is not associated with hypothyroidism (7), in fact 2 of 14 patients had elevated peak TSH levels after TRH stimulation. It is interesting to note that the TSH assay used in that study was an older, less sensitive assay with an upper normal limit of 8 µIU/ml. The mean basal TSH in the PHP1B group was 4.5 compared with 2.5 in the normal group, and therefore, the patients were reported to have normal TSH levels despite the fact that the levels tended to be higher than those in normal subjects. It would be interesting to know how many patients in the published cohort would have been reported to have had mildly elevated TSH levels if more sensitive TSH assays were available. More recently, a patient with paternal uniparental disomy of 20q, which leads to the GNAS imprinting pattern present in PHP1B, developed both PTH resistance and hypothyroidism (45). Two PHP1B kindreds reported by another group had hypothyroidism in several family members, but the hypothyroidism did not necessarily cosegregate with PHP1B, suggesting that the hypothyroidism is due to an unrelated cause, such as autoimmunity (46, 47).
It should be emphasized that we did not directly measure Gs expression in the thyroid glands of these patients. The levels of thyroid-specific Gs expression shown for PHP1A, PPHP, and PHP1B in Fig. 3 do not take into account the possibility that the actual levels of Gs expression may be altered by compensatory mechanisms. Also, we have just shown a potential mean value for Gs expression, but in reality the severity of Gs deficiency (and therefore TSH resistance) will vary between patients due to the normal variation in thyroid-specific Gs imprinting within the population. PHP1B patients who show evidence for TSH resistance may have greater suppression of paternal-specific Gs expression than those without TSH resistance. Given the fact that PHP1A and PHP1B patients have renal PTH resistance of similar severity (7), it seems likely that allele-specific Gs expression in renal proximal tubules is even more skewed toward the maternal allele than in the thyroid.
In summary, we have shown that Gs is imprinted in human thyroid glands and that this appears to be important in the development of moderate TSH resistance in PHP1A (but not PPHP) and less severe TSH resistance in some, but not all, PHP1B patients. In making the correct diagnosis of patients who present with renal PTH resistance, patients who have normal TSH most likely have PHP1B, whereas those with elevated levels may have either PHP1A or PHP1B. TSH levels greater than 10 µIU/ml are more consistent with PHP1A, whereas levels less than 10 µIU/ml do not help differentiate between PHP1A and PHP1B.
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Footnotes |
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Abbreviations: AHO, Albright hereditary osteodystrophy; PHP1A, pseudohypoparathyroidism type 1A; PPHP, pseudopseudohypoparathyroidism.
Received March 7, 2003.
Accepted June 2, 2003.
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 M. Chen, O. Gavrilova, J. Liu, T. Xie, C. Deng, A. T. Nguyen, L. M. Nackers, J. Lorenzo, L. Shen, and L. S. Weinstein Alternative Gnas gene products have opposite effects on glucose and lipid metabolism PNAS, May 17, 2005; 102(20): 7386 - 7391. [Abstract] [Full Text] [PDF]
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J. Liu, M. Chen, C. Deng, D. Bourc'his, J. G. Nealon, B. Erlichman, T. H. Bestor, and L. S. Weinstein From the Cover: Identification of the control region for tissue-specific imprinting of the stimulatory G protein {alpha}-subunit PNAS, April 12, 2005; 102(15): 5513 - 5518. [Abstract] [Full Text] [PDF]
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