Gs activity is reduced in erythrocyte membranes ... - Wiley Online Library

JBMR

ORIGINAL ARTICLE

Gsa Activity Is Reduced in Erythrocyte Membranes of Patients With Psedohypoparathyroidism Due to Epigenetic Alterations at the GNAS Locus Celia Zazo , 1 Susanne Thiele , 2 Cesar Martıń , 3 Eduardo Fernandez-Rebollo , 4 Lorea Martinez-Indart , 5 Ralf Werner , 2 Intza Garin , 1 Spanish PHP Group , 1 Olaf Hiort , 2 and Guiomar Perez de Nanclares 1 1

Molecular Genetics Laboratory, Research Unit, Hospital Txagorritxu, Vitoria-Gasteiz, Spain Department of Pediatric and Adolescent Medicine, University of Lu¨beck, Germany 3 Unidad de Biofı´sica, Departamento de Bioquı´mica, Universidad del Paı´s Vasco, Centro Mixto CSIC-UPV/EHU, Bilbao, Spain 4 Endocrine Unit, Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, USA 5 Clinical Epidemiology Unit, Research Unit, CAIBER-Hospital de Cruces, Barakaldo-Bizkaia, Spain 2

ABSTRACT In pseudohypoparathyroidism (PHP), PTH resistance results from impairment of signal transduction of G protein–coupled receptors caused by a deficiency of the Gsa-cAMP signaling cascade due to diminished Gsa activity in maternally imprinted tissues. In PHP-Ia, inactivating mutations of the GNAS gene lead to haploinsufficiency in some tissues with biallelic expression, so in addition to PHP, Albright’s hereditary osteodystrophy (AHO) is also present. In PHP-Ib, caused by methylation defects at the GNAS locus, diminished Gsa activity was thought to be limited to maternally imprinted tissues, such as the renal proximal tubule and the thyroid, leading to a lack of AHO. Recently, we demonstrated methylation defects in patients with AHO signs, indicating a connection between epigenetic changes and AHO. Our objective was to determine Gsa activity in erythrocyte membranes in patients with epigenetic defects at the GNAS locus compared to normal controls and patients with inactivating GNAS mutations. Gsa activity and expression, mutation of the GNAS locus, and methylation status were studied in patients with PHP and mild signs of AHO (PHP-Ia: 12; PHP-Ib: 17, of which 8 had some features of AHO). Then, we statistically compared the Gsa activity of the different PHP subtypes. Patients with methylation defects at the GNAS locus show a significant decrease in erythrocyte Gsa activity compared to normal controls (PHP-Ib versus controls, p < .001). This was significantly lower in patients with AHO signs (PHP-Ib þ mild-AHO versus PHP-Ib, p < .05). Our research shows that PHP-Ia and PHP-Ib classification is not only overlapped genetically, as reported, but also in terms of Gsa activity. Reduced expression of GNAS due to methylation defects could downregulate Gsa activity in other tissues beyond those described and could also be causative of AHO. ß 2011 American Society for Bone and Mineral Research. KEY WORDS: PSEUDOHYPOPARATHYROIDISM; Gsa PROTEIN; GNAS; METHYLATION DEFECTS; AHO PHENOTYPE

Introduction

T

he stimulatory G protein alpha subunit (Gsa), the main product coded by he GNAS gene locus in region 20q13, is crucial for cAMP-mediated signalling pathways. Gsa, together with the b and g subunits, forms the heterotrimeric G protein. G proteins are GTP-dependent complexes that release Gsa to catalyse the replacement of GDP by GTP after the ligand interaction with the G protein coupling receptor (GPCR) occurs. This acts as the driving force enabling the unidirectionality of the Gsa cycle to be maintained.(1)

Apart from Gsa, the GNAS locus gives rise to different products through the use of alternative promoters and first exons that splice onto a common set of downstream exons. These additional gene products are oppositely imprinted: XLas, NESPas and A/B are paternally expressed and maternally silenced by methylation, whereas NESP55 is maternally expressed and paternally silenced.(2,3) Inactivating mutations of the maternal allele in the Gsaencoding exons (summarized in www.hgmd.cf.ac.uk) and epigenetic changes(3) have been described in the literature as a cause of a Gsa-mediated hormonal disorder, which is

Received in original form November 8, 2010; revised form January 14, 2011; accepted February 9, 2011. Published online February 23, 2011. Address correspondence to: Guiomar Perez de Nanclares, PhD, Molecular Genetics Lab Research Unit, Hospital Txagorritxu, 01009, Vitoria-Gasteiz, Alava, Spain. E-mail: [email protected] Journal of Bone and Mineral Research, Vol. 26, No. 8, August 2011, pp 1864–1870 DOI: 10.1002/jbmr.369 ß 2011 American Society for Bone and Mineral Research

1864

characterized by PTH renal resistance and is known as pseudohypoparathyroidism (PHP). Based on clinical signs, findings from laboratory tests, and the results of an in vitro assay measuring the Gsa protein activity in erythrocyte membranes from patients, PHP is divided into several subtypes.(4,5) PHP-Ia (OMIM: 103580) is clinically characterized by multihormonal resistance and Albright’s hereditary osteodystrophy (AHO; brachymetacarpia, brachymetatarsia, obesity, short stature, subcutaneous ossifications, and mental retardation)(6) and is commonly caused by inactivating GNAS mutations. Although Gsa is expressed biallelically in most tissues (including blood cells), in the pituitary, thyroid, and gonads it is only maternally expressed and paternally silenced by imprinting.(7,8) Thus, only maternal transmission of GNAS mutations leads to PHP-Ia, whereas the paternal inheritance of the same mutations leads to pseudo-pseudohypoparathyroidism (PPHP), with the presence of AHO but the absence of hormone resistance.(4) In contrast, people who have PHP-Ib (OMIM: 603233) present with renal PTH and partial TSH resistance, usually in the absence of the AHO phenotype and resistance to other hormones.(9) PHPIb is associated with a loss of methylation (LOM) at the GNAS exon A/B (also called 1A), sometimes combined with epigenetic defects at other GNAS differentially methylated regions. The familial form of the disease has been shown to be mostly associated with a limited exon A/B methylation defect and a heterozygous 3-kb or 4.4-kb deletion mutation within the closely linked STX16 gene.(10,11) The exon A/B region is known as an imprinting control region and is believed to be critical for the tissue-specific imprinting of Gsa in the renal proximal tubules.(2) Methylation changes in the NESP55, NESPas, and XLas promoter regions have only been described in patients with a sporadic form of PHPIb,(12) except for in a few families with an additional NESPas deletions.(13,14) In a previous paper, we described for the first time five patients with PHP and mild AHO features associated with methylation defects at the GNAS locus.(15) Our data indicated slightly reduced levels of Gsa activity in the erythrocyte membranes of these patients, a finding that was also reported by Freson et al. in the platelets of patients with PHP-Ib.(16) Thus, the aim of the current study was to investigate the activity of Gsa solubilized from erythrocyte membranes in a larger cohort of PHP patients with changes in the methylation status of the GNAS gene locus (with or without mild AHO signs), to confirm the influence of epigenetic changes in Gsa activity in tissues where Gsa expression is assumed to be biallelic.

Patients and Methods Cases The patient population was selected according to the following diagnostic parameters: elevated PTH levels, hypocalcaemia, and hyperphosphataemia in the absence of vitamin D deficiency.(6) Some patients had, in addition, clinical signs of AHO. Coding mutations within the GNAS gene were excluded according to previously described methods.(15) We included as positive Gsa ACTIVITY IN PHP PATIENTS WITH EPIGENETIC DEFECTS

controls PHP-Ia patients with signs of AHO and PHP, caused by inactivating mutations of the Gsa-encoding exons of GNAS. Studies were approved by the ethical committee of Cruces Hospital, Txagorritxu Hospital, and the University of Lu¨beck.

Methylation analysis of the GNAS cluster by MS-MLPA Dosage and methylation analyses on leukocyte DNA were carried out by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) using the ME031A kit (MRC-Holland, Amsterdam, The Netherlands) as previously described.(17) Analysis of the MS-MLPA PCR products was performed on an ABI3500 genetic analyzer using GeneMapper software (Applied Biosystems, Foster City, CA, USA).

STX16 and NESP55 deletion analysis The presence of the 3-kb and 4.4-kb deletion mutations within STX16 in patients with isolated methylation defects at exon A/B was confirmed by PCR as previously described.(10,13) PHP-Ib patients with an overall GNAS methylation defect were screened for NESPas deletions by PCR.(13)

Gsa activity measurement In heparinized blood samples, the activity of Gsa protein from erythrocyte membranes of the patients was analyzed in vitro using an adapted version of the method of Levine et al.(18) as previously described.(5) Briefly, after solubilization and activation of the Gsa with GTPgS (guanosine 5’-[g-thio]triphosphate), the generation of cAMP through adenylyl cyclase from turkey red cell membranes was measured in the presence of ATP by RIA (IBL International, Hamburg, Germany). Results obtained in triplicate were expressed as a percentage of the mean found in healthy volunteers (normal range: 85–115%). Normal control samples were taken from 11 healthy adults. These samples were obtained and processed at the same time as those from the patients.

Immunoblot analysis Solubilized membranes from a representative group of patients were available for Gsa protein quantification using Western blot analysis. Protein fractions (30 mg total) were separated electrophoretically on an 8.5% SDS polyacrylamide gel and transferred to nitrocellulose membrane in Trans-Blot SD equipment (BioRad, Hercules, CA, USA). Blots were blocked overnight at 48C with 10% powdered skimmed milk in TBST buffer (Tris 10 mM, NaCl 150 mM, Tween-20 0.05% [p/v], pH 7.5). Incubation with primary antibodies (2 hours at room temperature) and secondary antibodies (1 hour at room temperature) was performed in TBST buffer with 2.5% (p/v) milk. Proteins were detected using an enhanced chemiluminescence detection system (Amersham ECL, GE Healthcare, Piscataway, NJ, USA). Blots were revealed with anti-Gsa mouse monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-flotillin 1 rabbit polyclonal antibody (Abcam, Cambridge, MA, USA), anti-mouse IgG HRPlinked antibody (Cell Signaling Technology, Danvers, MA, USA) and anti-rabbit IgG HRP-linked antibody (Cell Signaling Technology). The Quantity One Image Analyzer software program (Bio-Rad) was used for quantitative densitometric analysis. Journal of Bone and Mineral Research

1865

Statistical analysis

Genetic and epigenetic results

Pairwise comparisons were made between each group of patients and controls, and the statistical significance of observed differences was determined using the Mann-Whitney U test (GraphPad Software, San Diego, CA, USA).

We excluded mutations in the Gsa-encoding exons of GNAS in patients 1 through 17 and identified five different heterozygous mutations in PHP-Ia patients. The GNAS mutation in patient 19 is a de novo mutation, whereas patients 20 through 29 had inherited the mutation from their mothers. Parental DNA samples were not available for patient 18. The methylation status of the exon A/B, NESP55, and XLas regions of the GNAS cluster was studied. Patients 1 through 6 had an isolated exon A/B epigenetic defect associated with the 3-kb STX16 deletion; the parental origin of these STX16 deletions was determined when samples were available. There were overall imprinting defects in patients 7 through 17, but, we did not detect a deletion in the NESP55/NESPas region in these individuals.

Results Patients Table 1 summarizes the clinical data of 29 cases, comprising 17 unrelated patients with a clinical diagnosis of PHP-Ib (8 of them with mild AHO features) and 12 patients with a clinical diagnosis of PHP-Ia, AHO features, and proven mutations in GNAS. Some of the patients have been described previously.(15,17,19)

Table 1. Clinical, Genetic, and Biochemical Features of the Patient Cohort at Diagnosis

Patient no. Sex

Clinical classification

1 2 3 4 5[15] 6[15] 7[15]

F PHP-Ib M PHP-Ib F PHP-Ib F PHP-Ib; mild-AHO (Br, RF) F PHP-Ib; mild-AHO (Br, RF) F PHP-Ib; mild-AHO (Br, RF, Ob) F PHP-Ib; mild-AHO (Br, RF)

8[15] 9 10 11 12 13 14 15 16[17] 17[17]

F M F F F M F F F M

18 19 20[17] 21[17] 22[17] 23[19] 24 25 26 27 28 29

F F F M M F F F M F F M

P PTH Ca (pg/mL) (mg/dL) (mg/dL) NV: see Gsa NV: NV: Age (years) 40–65 8.1–10.4 legend activity 23 11 9.83 29 22 23 31

630 410 290 386 180 355 102

5.3 5.9 6.8 7.2 6.9 9.02 5.78

PHP-Ib; mild-AHO (Br, RF) 19 PHP-Ib; mild-AHO (Br, RF, MR) 5.9 PHP-Ib; mild-AHO (Br, RF) 6 PHP-Ib; mild-AHO (Br, RF) 12 PHP-Ib 19 PHP-Ib 14.08 PHP-Ib 14 PHP-Ib 8 PHP-Ib 23 PHP-Ib 9

500 120 454 284 157 479 209 569 224 940

6.3 5.6 7.5 6.4 6.5 5.4 4.8 6 6.9 8.4

5.4 8.6 8.9 6.2 7.5 7.6 10 10.5 5 6.5

71.7 85 68.6 70.7 100.1 96.5 96.5 92 92 81

155 70 113 633 434 264 495 215 549 585 484 279

7.6 10.7 5.6 5.6 5.1 9.7 7.7 5.8 6.8 9.1 4.6 6.5

5 6.2 4.2 7.9 10.3 7 6.3 11.5 8.5 2.2 3.4 9.3

57.8 59.9 59.6 59.4 57.4 62 43.5 49.2 71.1 56 55.6 49

PHP-Ia PHP-Ia PHP-Ia PHP-Ia PHP-Ia PHP-Ia PHP-Ia PHP-Ia PHP-Ia PHP-Ia PHP-Ia PHP-Ia

28 1 19 10 3 3 2.25 4 2.67 2.83 2.58 2.58

6.5 8.5 8.7 3.8 5.58 4.1 4.02

88.2 88.1 89.7 54.7 95.5 81.2 93.75

Genetic origin

GNAS alteration A/B LOM (STX16 3kb deletion) A/B LOM (STX16 3kb deletion) A/B LOM (STX16 3kb deletion) A/B LOM (STX16 3kb deletion) A/B LOM (STX16 3kb deletion) A/B LOM (STX16 3kb deletion) NESPAS, A/B LOM, Xlas partial LOM NESPAS, Xlas, A/B LOM NESPAS, Xlas, A/B LOM NESPAS, Xlas, A/B LOM NESPAS, Xlas, A/B LOM NESPAS, Xlas, A/B LOM NESPAS, XLas, A/B LOM NESPAS, XLas, A/B LOM NESPAS, Xlas, A/B LOM NESPAS, Xlas, A/B LOM Pat20UPD

Maternal Maternal NA NA Maternal Maternal

c.91C > T; p.Q31X c.91C > T; p.Q31X c.971-1G > A c.971-1G > A c.565_568delGACT; p.Y190MfsX14 g.56,883,365_56,913,796del c.565_568delGACT; p.Y190MfsX14 c.565_568delGACT; p.Y190MfsX14 c.565_568delGACT; p.Y190MfsX14 c.565_568delGACT; p.Y190MfsX14 c.565_568delGACT; p.Y190MfsX14 c.565_568delGACT; p.Y190MfsX14

NA De novo Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal Maternal

The thin line divides patients with epigenetic defects from those who carry a mutation. NA: not available; LOM: loss of methylation. F, Female; M, male; Br, brachydactyly, clinically evident; Br, radiological evidence of brachydactyly; RF, round face; Ob, obesity defined as body mass index above 30 kg/m2 in adults or weight above the 97th percentile in children; MR: mental retardation; NV: normal value. Phosphorus normal values: 3.8–6.5 mg/dL (1–3 yrs), 3.7–5.6 mg/dL (4–11 yrs), 2.9–5.4 mg/dL (12–15 yrs) and 2.4–4.0 mg/dL (>15 yrs).

1866

Journal of Bone and Mineral Research

ZAZO ET AL.

After their genetic and epigenetic characterization, patients were grouped into three clusters: patients with inactivating mutations in the Gsa-encoding exons of GNAS, patients with methylation defects (with and without AHO signs), and controls.

Gsa activity measurement To test whether our Gsa measurement was valid, we analyzed, as positive controls, PHP-Ia patients carrying protein-truncating mutations. Because GNAS does present biallelic expression in erythrocyte membranes, we would expect a loss of activity of about 50%. We confirm that Gsa functionality was impaired in the erythrocyte membranes of these affected patients (mean: 56.71% versus 102.45% in tested controls). When analyzing patients with methylation defects (LOM), we found that the Gsa activity was 85.01%, 17.5% less than in controls. Comparing the Gsa activity for patients with mutations, patients with methylation defects, and controls, we found strongly statistically significant differences among all the clusters ( p < .001) (Fig. 1A). Moreover, Gsa activity was significantly lower in patients with LOM and signs of AHO than in those without AHO signs (77.64% versus 91.57%, respectively; p < .05; Fig. 1B). There were no statistically significant differences in Gsa activity in patients with overall methylation defects (mean: 86.17%, SD: 11.48) compared with patients who had isolated exon A/B methylation defects (mean: 82.9%, SD: 14.54) (Fig. 1C).

Immunoblot analysis Patients with epigenetic defects and signs of AHO had lower levels of Gsa protein expression in membranes than those with no manifestations of AHO (0.43 0.04 versus 0.61 0.07, p ¼ .05). There were no statistically significant differences in Gsa expression between patients with overall methylation defects and those with isolated exon A/B methylation defects (0.54 0.09 and 0.53 0.14, respectively) (Fig. 2).

Discussion Until recently, it was believed that only a mutation in the Gsaencoding exons of GNAS would lead to a haploinsufficiency of Gsa in some tissues with biallelic expression causing AHO. However, a loss of methylation at the maternally imprinted exon A/B would influence only Gsa protein activity in imprinted tissues but lead to normal Gsa activity in biallelic expressed tissues, such as erythrocyte membranes,(2) leading to PHP but not to AHO. Further, it was thought that the Gsa-encoding gene, GNAS, was biallelically expressed in most tissues and paternally silenced in some endocrine tissues, such as renal proximal tubules, pituitary, thyroid, and gonads. Against this background, in 2007 our group described a series of five patients clinically classified as PHP-Ia because of the presence of a mild AHO phenotype and PTH and TSH resistance that carried epigenetic alterations at the GNAS locus.(15) This observation has been corroborated by independent research teams.(20–22) Given this, we hypothesized that if the AHO-like features were related to GNAS imprinting defects, alteration in GNAS imprinting may contribute, at least in some patients, to the pathogenesis of AHO. Gsa ACTIVITY IN PHP PATIENTS WITH EPIGENETIC DEFECTS

Fig. 1. Gsa activity levels in (A) patients with mutations in the GNAS gene (n ¼ 12), patients with methylation defects (LOM) (n ¼ 17) and controls (n ¼ 11); (B) patients with LOM with (n ¼ 8) versus without (n ¼ 9) an AHO phenotype and (C) patients with isolated exon A/B methylation defects (n ¼ 6) versus those with overall LOM (n ¼ 10). The thick horizontal lines correspond to the median values, the gray rectangles span the 25th to 75th percentiles, and the error bars indicate the range of the standard deviations. LOM: loss of methylation.


1867

Fig. 2. Gsa expression in erythrocytes. Immunoblot analysis (A) and densitometric scanning (B) of Gsa and flotillin proteins in erythrocyte lysates from PHP-Ia patients with mutations at GNAS gene, mild AHO patients with an exon 1A–only methylation defect, mild AHO patients with an overall methylation defect, PHP-Ib patients with an exon 1A–only methylation defect, and PHP-Ib patients with overall methylation defect. The results are expressed as the densitometric ratio of Gsa and flotillin for each patient.

In the current study, we have demonstrated for the first time a statistically significant reduction in Gsa activity in erythrocyte membranes in a large cohort of patients with methylation defects at the GNAS gene locus. Further, when we compare Gsa activity in patients who have epigenetic defects with and without AHO characteristics, we find significant differences: the activity is lower in patients who do present the phenotype, indicating that a Gsa activity ‘‘threshold’’ is needed for a normal phenotype. So, we confirm that AHO features present in some patients with epigenetic changes at the GNAS locus are due to Gsa imprinting. Consistent with recent evidence that obesity is a direct consequence of Gsa imprinting in one or more specific tissues,(23–25) this interpretation implies that Gsa expression is subject to allelic bias in a larger number of tissues than is currently recognized and that the degree of this allelic bias varies among individuals.(15,22) Indeed, a previous report has demonstrated modest maternal predominance of Gsa expression in 1 of 19 human bone samples,(26) which could also explain the AHO phenotype in some patients. A reduced level of Gsa activity was also described by Freson et al. in a subgroup of patients that had been clinically classified as PHP-Ib using the platelet aggregation-inhibition test.(16) They reported that PHP-Ib patients with an overall methylation defect presented lower levels of Gsa and increased XLas protein levels, leading to a decrease in Gsa activity. Nevertheless, this was not observed in those patients with an isolated A/B methylation defect. We wanted to confirm these results in our cohort of patients with methylation defects at the GNAS locus by measuring Gsa activity in erythrocyte membranes because as ours is a multicenter project, it proved too difficult to receive and process the blood samples in less than 4 hours, as is required for the platelet aggregation-inhibition test. Besides, the alternative method we employed is commonly used achieving a correct diagnosis of patients with pseudohypoparathyroidism.(18) We did not find any significant differences in Gsa activity between patients with isolated exon A/B methylation defects

1868


and patients with an overall methylation defect. This is in accordance with the clinical findings of Linglart et al., demonstrating that individuals with overall versus those with limited exon A/B methylation defects have similar clinical and laboratory features.(27) It is important to ascertain whether different methods used (in particular, a complementation assay with turkey erythrocyte membranes versus the platelet aggregationinhibition test) could have an influence on these results. The lower level of Gsa activity in PHP-Ib patients found by Freson and in our work suggests that the isolated exon A/B methylation defect reduces GNAS expression, as previously postulated(2) and proved by our results; this protein reduction is, in turn, responsible for the decrease in Gsa activity. On the other hand, further studies are needed to determine whether the XLas overexpression, due to an overall methylation defect, downregulates Gsa expression leading to Gsa hypofunction, as Freson described in platelets.(16) Overall, a reduced level of expression of GNAS due to methylation defects seems to downregulate Gsa activity in other tissues beyond the kidney and the thyroid and, thus, could also be a cause of AHO. These results support the idea that PHP-Ia and PHP-Ib classifications overlap, as noted by us and others,(15,20–22) not only from genetic point of view but also in terms of Gsa activity. It seems that only multiple hormone resistance as a characteristic of PHP-Ia(28–31) can be clinically used to distinguish it from PHP-Ib.(9,32) We propose that a new classification should be considered in which the knowledge about genetic and epigenetic data is included and not just reduced to the clinical information.

Disclosures All the authors state that they have no conflicts of interest.

Acknowledgments C Zazo and S Thiele contributed equally to this work. We thank all family members for their participation and their physicians for sending us the samples. The authors would like to thank Valeria Romanelli for her helpful comments regarding the interpretation of the MS-MLPA results. GPN receives funding from the Carlos III Health Institute I3SNS Programme (CP03/0064; SIVI 1395/09). This work was partially supported by grants GV2008/111035 from the Basque Department of Health, BIO08/ER/001, and the Eugenio Rodriguez Pascual foundation (awarded to GPN), as well as grants from the German Ministry for Research and Education (BMBF No: GMG 01GM0315, awarded to OH, RW, and ST). This group is founded by the Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER), ISCIII. Members of the Spanish PHP Group: Complejo Hospitalario de Caćeres, Caćeres (Arroyo J.); Complejo Hospitalario Dr. Negrin, Las Palmas de Gran Canaria (Saéz F., Sańchez A.); Complejo Hospitalario Materno Insular, Las Palmas de Gran Canaria (Dom´ınguez A., Santana A.); Complejo Hospitalario Universitario de Albacete, Albacete (Ruiz R.); Complexo Hospitalario Universitario Santiago de Compostela, A Corunã (Castro L.); Hospital Central de ZAZO ET AL.

Asturias, Asturias (Rivas C.); Hospital Clıńico San Carlos, Madrid (Pe´rez O.); Hospital de Barbanza, A Corunã (Molinos S.), Hospital de Cruces, Bizkaia (Castanõ L., Gaztambide S., Moure MD., Vela A.); Hospital de Donostia, Gipuzkoa (Saez R., Unanue G.); Hospital de Navarra, Navarra (Meneńdez E., Anda A.); Hospital de Nens, Barcelona (Pavia C.); Hospital de Txagorritxu, A´lava (Diez-Lopez I.); Hospital del Mar, Barcelona (Bonet M.); Hospital Do Meixoeiro, Vigo (Morales M.J.); Hospital General de Alicante, Alicante (Zapico M.); Hospital General de Ciudad Real, Ciudad Real (Aguirre M.); Hospital Infantil Universitario del Ninõ Jesu´s, Madrid (Munõz MT., Rubio-Cabezas O., Argente J.); Hospital Infantil Vall D´Hebron, Barcelona (Audi L., Yeste D.); Hospital Materno Infantil Carlos Haya, Ma´laga (Soriguer F.); Hospital Prıńcipe de Viana, Navarra (Garcıá M., Rodrı´guez R.M., Gon˜i M.J.); Hospital Puerta del Mar; Ca´diz (Armenta D., Gonzalez-Duarte D); Hospital Ramoń y Cajal, Madrid (Barrio R.); Hospital San Jorge, Huesca (Ca´mara A.); Hospital Sant Joan de Deú, Barcelona (Martorell L., Sua´rez L., Cardona R., Gean E); Hospital Severo Ochoa, Madrid (GarcıáCuartero B.); Hospital Teresa de Herrera, A Corunã (Pereira M.S., Rodrı´guez B.); Hospital Universitario 12 de Octubre, Madrid (Azriel S.); Hospital Universitario de Guadalajara, Guadalajara (Jimeńez J.M, Sentchordi L.); Hospital Universitario de Valme, Sevilla (Espino-Aguilar R.); Hospital Universitario La Fe, Valencia (Beneyto M.); Hospital Universitario La Paz, Madrid (A´lvarez C., Lecumberri B.); Hospital Universitario Marques de Valdecilla, Cantabria (Luzuriaga C.); Hospital Universitario Miguel Servet, Zaragoza Calvo MT., Labarta JI.); Hospital Universitario Prıńcipe de Asturias, Madrid (Saavedra P.); Hospital Universitario Reina Sofıá, Co´rdoba (Can˜ete Estrada R.); Hospital Universitario San Cecilio, Granada (Ordunã R.); Hospital Universitario Virgen de la Arriaxaca, Murcia (Guillen-Navarro E., Guillen C., Gon˜i F.); Hospital Universitario Virgen del Rocıó, Sevilla (Del Valle J.); Hospital Virgen de la Salud, Toledo (Luque I., Meneńdez A.); Hospital Virgen del Camino, Navarra (Oyarzabal M.)

References 1. Wettschureck N, Offermanns S. Mammalian G proteins and their cell type specific functions. Physiol Rev. 2005;85:1159–1204. 2. Weinstein LS, Yu S, Warner DR, Liu J. Endocrine manifestations of stimulatory G protein alpha-subunit mutations and the role of genomic imprinting. Endocr Rev. 2001;22:675–705. 3. Bastepe M. The GNAS locus and pseudohypoparathyroidism. Adv Exp Med Biol. 2008;626:27–40. 4. Lania A, Mantovani G, Spada A. G protein mutations in endocrine diseases. Eur J Endocrinol. 2001;145:543–559. 5. Ahrens W, Hiort O, Staedt P, Kirschner T, Marschke C, Kruse K. Analysis of the GNAS1 gene in Albright’s hereditary osteodystrophy. J Clin Endocrinol Metab. 2001;86:4630–4634. 6. Albright F, Burnett CH, Smith PH, Parson W. Pseudohypoparathyroidsm: an example of ‘‘Seabright syndrome.’’ Endocrinology. 1942;30: 922–932. 7. Mantovani G, Ballare E, Giammona E, Beck-Peccoz P, Spada A. The gsalpha gene: predominant maternal origin of transcription in human thyroid gland and gonads. J Clin Endocrinol Metab. 2002; 87:4736–4740. 8. Hayward BE, Barlier A, Korbonits M, et al. Imprinting of the G(s)alpha gene GNAS1 in the pathogenesis of acromegaly. J Clin Invest. 2001; 107:R31–R36.

Gsa ACTIVITY IN PHP PATIENTS WITH EPIGENETIC DEFECTS

9. Liu J, Erlichman B, Weinstein LS. The stimulatory G protein alphasubunit Gs alpha is imprinted in human thyroid glands: implications for thyroid function in pseudohypoparathyroidism types 1A and 1B. J Clin Endocrinol Metab. 2003;88:4336–4341. 10. Linglart A, Gensure RC, Olney RC, Juppner H, Bastepe M. A novel STX16 deletion in autosomal dominant pseudohypoparathyroidism type Ib redefines the boundaries of a cis-acting imprinting control element of GNAS. Am J Hum Genet. 2005;76:804–814. 11. Bastepe M, Frohlich LF, Hendy GN, et al. Autosomal dominant pseudohypoparathyroidism type Ib is associated with a heterozygous microdeletion that likely disrupts a putative imprinting control element of GNAS. J Clin Invest. 2003;112:1255–1263. 12. Liu J, Nealon JG, Weinstein LS. Distinct patterns of abnormal GNAS imprinting in familial and sporadic pseudohypoparathyroidism type IB. Hum Mol Genet. 2005;14:95–102. 13. Bastepe M, Frohlich LF, Linglart A, et al. Deletion of the NESP55 differentially methylated region causes loss of maternal GNAS imprints and pseudohypoparathyroidism type Ib. Nat Genet. 2005; 37:25–27. 14. Chillambhi S, Turan S, Hwang DY, Chen HC, Juppner H, Bastepe M. Deletion of the noncoding GNAS antisense transcript causes pseudohypoparathyroidism type Ib and biparental defects of GNAS methylation in cis. J Clin Endocrinol Metab. 2010;95:3993–4002. 15. de Nanclares GP, Fernandez-Rebollo E, Santin I, et al. Epigenetic defects of GNAS in patients with pseudohypoparathyroidism and mild features of Albright’s hereditary osteodystrophy. J Clin Endocrinol Metab. 2007;92:2370–2373. 16. Freson K, Izzi B, Labarque V, et al. GNAS defects identified by stimulatory G protein alpha subunit signalling studies in platelets. J Clin Endocrinol Metab. 2008;93:4851–4859. 17. Lecumberri B, Fernandez-Rebollo E. Coexistence of two different pseudohypoparathyroidism subtypes (Ia and Ib) in the same kindred with independent Gs{alpha} coding mutations and GNAS imprinting defects. J Med Genet. 2010;47:276–280. 18. Levine MA, Downs RW Jr, Singer M, Marx SJ, Aurbach GD, Spiegel AM. Deficient activity of guanine nucleotide regulatory protein in erythrocytes from patients with pseudohypoparathyroidism. Biochem Biophys Res Commun. 1980;94:1319–1324. 19. Fernandez-Rebollo E, Garcia-Cuartero B, Garin I, et al. Intragenic GNAS deletion involving exon A/B in pseudohypoparathyroidism type 1A resulting in an apparent loss of exon A/B methylation: potential for misdiagnosis of pseudohypoparathyroidism type 1B. J Clin Endocrinol Metab. 2010;95:765–771. 20. Mariot V, Maupetit-Mehouas S, Sinding C, Kottler ML, Linglart A. A maternal epimutation of GNAS leads to Albright osteodystrophy and PTH resistance. J Clin Endocrinol Metab. 2008;93:661–665. 21. Unluturk U, Harmanci A, Babaoglu M, et al. Molecular diagnosis and clinical characterization of pseudohypoparathyroidism type-Ib in a patient with mild Albright’s hereditary osteodystrophy-like features, epileptic seizures, and defective renal handling of uric acid. Am J Med Sci. 2008;336:84–90. 22. Mantovani G, de Sanctis L, Barbieri AM, et al. Pseudohypoparathyroidism and GNAS epigenetic defects: clinical evaluation of albright hereditary osteodystrophy and molecular analysis in 40 patients. J Clin Endocrinol Metab. 2010;95:651–658. 23. Long DN, McGuire S, Levine MA, Weinstein LS, Germain-Lee EL. Body mass index differences in pseudohypoparathyroidism type 1a versus pseudopseudohypoparathyroidism may implicate paternal imprinting of Galpha(s) in the development of human obesity. J Clin Endocrinol Metab. 2007;92:1073–1079. 24. Xie T, Chen M, Gavrilova O, Lai EW, Liu J, Weinstein LS. Severe obesity and insulin resistance due to deletion of the maternal Gsalpha allele is reversed by paternal deletion of the Gsalpha imprint control region. Endocrinology. 2008;149:2443–2450.


1869

25. Chen M, Gavrilova O, Liu J, et al. Alternative Gnas gene products have opposite effects on glucose and lipid metabolism. Proc Natl Acad Sci U S A. 2005;102:7386–7391. 26. Mantovani G, Bondioni S, Locatelli M, et al. Biallelic expression of the Gsalpha gene in human bone and adipose tissue. J Clin Endocrinol Metab. 2004;89:6316–6319. 27. Linglart A, Bastepe M, Juppner H. Similar clinical and laboratory findings in patients with symptomatic autosomal dominant and sporadic pseudohypoparathyroidism type Ib despite different epigenetic changes at the GNAS locus. Clin Endocrinol (Oxf). 2007; 67: 822–831. 28. Mantovani G, Maghnie M, Weber G, et al. Growth hormone-releasing hormone resistance in pseudohypoparathyroidism type ia: new evidence for imprinting of the Gs alpha gene. J Clin Endocrinol Metab. 2003;88:4070–4074.

1870


29. Germain-Lee EL, Groman J, Crane JL, Jan de Beur SM, Levine MA. Growth hormone deficiency in pseudohypoparathyroidism type 1a: another manifestation of multihormone resistance. J Clin Endocrinol Metab. 2003;88:4059–4069. 30. Levine MA, Downs RW Jr, Moses AM, et al. Resistance to multiple hormones in patients with pseudohypoparathyroidism. Association with deficient activity of guanine nucleotide regulatory protein. Am J Med. 1983;74:545–556. 31. Wolfsdorf JI, Rosenfield RL, Fang VS, Kobayashi R, Razdan AK, Kim MH. Partial gonadotrophin-resistance in pseudohypoparathyroidism. Acta Endocrinol (Copenh). 1978;88:321–328. 32. Mantovani G, Bondioni S, Linglart A, et al. Genetic analysis and evaluation of resistance to thyrotropin and growth hormone-releasing hormone in pseudohypoparathyroidism type Ib. J Clin Endocrinol Metab. 2007;92:3738–3742.

ZAZO ET AL.