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Epigenetic Defects of GNAS in Patients with Pseudohypoparathyroidism and Mild Features of Albright’s Hereditary Osteodystrophy

Endocrinology and Diabetes Research Unit (G.P.d.N., E.F.-R., I.S., S.G., J.R.B., N.Z., L.C.), Hospital de Cruces, Barakaldo E48903, Basque Country, Spain; Pediatric Endocrinology Unit (B.G.-C.), Severo Ochoa Hospital, Leganés, 28911 Madrid, Spain; Departments of Medicine, Nursing, and Pediatrics (S.G., J.R.B., L.C.), University of Basque Country, 48940 Leioa, Bizkaia, Spain; Endocrinology Service (E.M.), Hospital de Navarra, 31080 Pamplona, Spain; Endocrinology Service (M.J.M.), Hospital do Meixoeiro, 36200 Viga, Pontevedra, Spain; Pediatric Endocrinology, Growth, and Adolescence Unit (M.P.), Clinical University of Santiago de Compostela, 15706 Santiago de Compostela, A Coruña, Spain; Fundación Pública Galega de Medicina Xenómica (F.B.), 15706 Santiago de Compostela, A Coruña, Spain; Department of Pediatric and Adolescent Medicine (W.A., O.H.), University of Lübeck, 23538 Lübeck, Germany; and Endocrine Unit (H.J., M.B.), Department of Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114

Address all correspondence and requests for reprints to: Luis Castaño, M.D., Ph.D., Endocrinology and Diabetes Research Group, Hospital de Cruces, Cruces-Barakaldo E48903 Bizkaia, Basque Country, Spain. E-mail: lcastano{at}hcru.osakidetza.net; or Murat Bastepe, M.D., Ph.D., Endocrine Unit, Massachusetts General Hospital, 50 Blossom Street, Thier 10, Boston, Massachusetts 02114. E-mail: bastepe{at}helix.mgh.harvard.edu.

	Abstract

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Context: Several endocrine disorders that share resistance to PTH are grouped under the term pseudohypoparathyroidism (PHP). PHP type I, associated with blunted PTH-induced nephrogenous cAMP formation and phosphate excretion, is subdivided according to the presence or absence of additional endocrine abnormalities, Albright’s hereditary osteodystrophy (AHO), and reduced Gs activity caused by GNAS mutations.

Objective: We sought to identify the molecular defect in four unrelated patients who were thought to have PHP-Ia because of PTH and TSH resistance and mild AHO features.

Methods: Gs activity and mutation analysis, and assessment of GNAS haplotype, methylation, and gene expression were performed for probands and family members.

Results: Two patients showed modest decreases in erythrocyte Gs activity. Instead of Gs point mutations, however, all four patients showed methylation defects of the GNAS locus, a feature previously described only for PHP-Ib. Furthermore, one patient with an isolated loss of GNAS exon A/B methylation had the 3-kb STX16 deletion frequently identified in PHP-Ib patients. In all but one of the remaining patients, haplotype analysis excluded large deletions or uniparental disomy as the cause of the observed methylation changes.

Conclusions: Our investigations indicate that an overlap may exist between molecular and clinical features of PHP-Ia and PHP-Ib. No current mechanisms can explain the AHO-like features of our patients, some of which may not be linked to GNAS. Therefore, patients with hormone resistance and AHO-like features in whom coding Gs mutations have been excluded should be evaluated for epigenetic alterations within GNAS.

	Introduction

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PSEUDOHYPOPARATHYROIDISM (PHP; MIM 103580) refers to end-organ resistance that primarily impairs the renal actions of PTH, a key regulator of calcium homeostasis that mediates its actions via a Gs-coupled receptor (1, 2). Patients with PHP-Ia show, besides PTH resistance, resistance to other hormones as well as Albright’s hereditary osteodystrophy (AHO), a constellation of features including short stature, obesity, brachydactyly, ectopic ossifications, and/or mental retardation. PHP-Ia is caused by maternally inherited, heterozygous inactivating mutations in one of the 13 GNAS exons encoding Gs. After paternal inheritance, the same mutations lead to pseudopseudohypoparathyroidism (PPHP), which refers to the presence of AHO without hormonal resistance.

Besides Gs, GNAS gives rise to several other mRNAs; these are derived from only one parental allele because of parent-specific methylation. XLs, A/B, and antisense are paternal, whereas NESP55 is maternal (1). In contrast, Gs is biallelic, except that in some tissues, such as renal cortex, paternal Gs expression is silenced.

Patients with PHP-Ib present with PTH and, occasionally, mild TSH resistance, but resistance to other hormones is usually absent (1, 2, 3). They also lack evidence of AHO and exhibit normal Gs activity in erythrocytes and fibroblasts. Nevertheless, genomic DNA from PHP-Ib patients show a loss of methylation at exon A/B, which is sometimes combined with epigenetic defects at other GNAS differentially methylated regions (4, 5, 6). Maternally inherited deletions within STX16 or NESP55, respectively, have been identified in these patients (7, 8, 9), leading to reduced Gs expression in the proximal tubule.

We now describe four unrelated cases who exhibited PTH and TSH resistance and physical and radiographic features consistent with mild AHO. Whereas these findings initially suggested PHP-Ia, molecular investigations revealed that each of these patients had abnormal GNAS methylation.

	Subjects and Methods

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Cases

Brachymetacarpia was established by the metacarpal sign (10) (cases 1, 3, and 4; Fig. 1) and/or z-score calculations of metacarpal length (11) (cases 1 and 2; Table 1). Upon diagnosis, each patient was treated with appropriate doses of calcium supplements, 1,25-dihydroxyvitamin D3, and levothyroxine.

View larger version (37K): [in this window] [in a new window]   FIG. 1. Hand x-rays of case 1 (A and B; at age 5 yr), case 2 (C and D), case 3 (E and F), and case 4 (G and H). The metacarpal sign was evident, except in case 2, because a line drawn through the distal ends of the fourth and fifth metacarpals crossed the third metacarpal (B, D, F, H). Note that case 2 exhibited short fourth metacarpals according to a z-score of –2 calculated by comparing metacarpal length with normative data.

Case 2. A 20-yr-old overweight woman (body mass index 26.3) was admitted after an apparent seizure. Her face was round; biochemical analyses revealed hypocalcemia, hyperphosphatemia, elevated serum PTH, and slightly elevated TSH with normal free T₄ (fT4) (Table 1); antithyroid peroxidase antibodies were elevated (797 IU). Calcium deposits were identified bilaterally by cranial computed tomography (CT) in frontal subcortical nuclei. Hand radiographs showed mild clinodactyly of both fifth fingers (Fig. 1, C and D) and shortening of fourth metacarpals (Table 1). She showed normal physical, mental, neurological, and reproductive development. Other family members had normal biochemical values.

Case 3. A 31-yr-old woman presented with short stature, round face, showed hypocalcemia, hyperphosphatemia, elevated serum PTH, low fT4, and elevated TSH (Table 1). Her hand radiographs showed slightly short fourth metacarpals (Fig. 1, E and F), and basal ganglia calcifications were noted on cranial CT. Neurological exam was unremarkable. She has two healthy children delivered after uneventful pregnancies.

Case 4. A 22-yr-old woman presented with an epileptic episode. Her laboratory studies showed hypocalcemia, hyperphosphatemia, elevated PTH, low fT4, and elevated TSH (Table 1); a CT revealed intracranial calcifications. She had a rounded face; her height and weight were within normal range. Radiographs revealed short fourth and fifth metacarpals of both hands (Fig. 1, G and H). She had previously been healthy and had regular menses. The patient’s 25-yr-old asymptomatic sister also showed elevated serum PTH with normal calcium and phosphorous; she also had a rounded face and her hand radiographs revealed slightly short fourth and fifth metacarpals (Table 1); no intracranial calcifications were detected. Both parents had normal blood biochemistries; the mother, but not the father, also showed slightly short fourth metacarpals.

Molecular studies

Mutational and methylation analysis. Genetic analyses were performed after informed consent had been obtained and upon approval by the institutional review board. The 13 Gs exons and GNAS exon A/B were amplified by PCR from blood leukocyte DNA and both strands were sequenced (primers and conditions are available on request).

Microsatellite typing, intragenic polymorphism analysis, and semiquantitative PCR were performed as previously described (12, 13). Semiquantitative amplification of noninformative markers was carried out, using an expressed sequence-tag fragment located on chromosome 5 as internal standard.

Exon A/B, NESP55, the antisense promoter, and XLs methylation was determined by PCR amplification after digestion of genomic DNA with methylation-sensitive and methylation-insensitive restriction enzymes and, independently, by combined bisulfite restriction analysis, as described (7, 13); FauI for A/B; AciI for NESP55; BstUI for XLs and the antisense promoter.

All DNA samples were screened for the 3-kb and 4.4-kb STX16 deletions by PCR, as described (7, 8). Genotyping at six polymorphic sites between nucleotides 3831 and 4712 (AL139349) within the STX16 region was performed, as described (7).

Gs activity measurement and RT-PCR

In heparinized blood samples, Gs activity in erythrocyte membranes was analyzed in vitro, as described (14, 15) [mean of healthy controls (range 85–115%)].

Quantitative gene expression analysis was performed by TaqMan real-time RT-PCR by using total RNA isolated from blood cells. Relative expression of each gene to ß-actin was calculated using the accurate cycle threshold method (16). Primers, probes, and conditions are available on request.

Gene dosage and Southern analysis

TaqMan real-time PCR was used to amplify the exon A/B genomic region and a region of the ß-actin gene (control) separately before and after digestion with the methylation-sensitive enzyme HpaII; no HpaII recognition sequences exist in the amplified portion of the ß-actin gene. For Southern analysis, a PCR-amplified, ³²P-labeled probe located 6.7-kb upstream of exon A/B was hybridized to BglII- or ScaI-digested genomic DNA blotted on nitrocellulose after separation on 0.5% agarose. Primers, probes, and conditions are available on request.

Statistical analysis

Statistical significance of observed differences between patients and controls were determined by using Mann-Whitney U statistic (GraphPad, San Diego, CA).

	Results

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Four patients showing evidence of PTH and TSH resistance were initially diagnosed with PHP-Ia because of phenotypic features suggestive of AHO, including a round face and mild shortening of fourth and/or fifth metacarpals (Table 1 and Fig. 1). Moreover, case 1 showed morbid obesity and ectopic ossifications. Case 2 was overweight and case 3 had short stature (Table 1). Consistent with the diagnosis of PHP-Ia, erythrocyte Gs activity in cases 1 and 2 was reduced. However, cases 3 and 4 appeared to have normal erythrocyte Gs activity (Table 1). Furthermore, the sequence analysis of the 13 GNAS exons encoding Gs failed to reveal nucleotide alterations, and the genotyping of patients and their parents (when available) revealed, except for case 1, at least one heterozygous polymorphism within the GNAS locus, ruling out a large deletion that comprises this gene (Table 1).

Because of the mild AHO findings and the lack of coding Gs mutations, we examined GNAS methylation, abnormalities of which are found in patients with PHP-Ib (4, 5). Analysis of the GNAS methylation status revealed loss of exon A/B methylation in each case, combined with additional methylation changes in cases 2 and 3; the affected sister of case 4 also demonstrated loss of exon A/B methylation (Table 1). Each patient, except for case 1, was heterozygous at the exon A/B pentanucleotide repeat (Table 1).

Because deletions of STX16 have been associated with isolated loss of exon A/B methylation (7, 8), we searched for the two previously reported STX16 microdeletions. This revealed, for case 4 and her affected sister, the presence of the heterozygous 3-kb STX16 microdeletion observed in AD-PHP-Ib (Table 1). The unaffected mother of case 4 also carried the same mutation (data not shown).

Whereas Gs mRNA in blood leukocytes did not differ significantly among the patients, PHP-Ia controls, and healthy controls, A/B mRNA appeared significantly higher in patient samples, compared with the control groups (Table 1), consistent with loss of exon A/B methylation (4).

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Our index cases showed, in addition to PTH and TSH resistance, phenotypic features that suggested mild AHO and were therefore diagnosed with PHP-Ia. Surprisingly, however, further investigations indicated that each case had molecular defects thought to be associated only with PHP-Ib.

Many of the individual AHO features are not specific to PHP (1, 2), and therefore, some or all of the AHO features in our patients may be unrelated to the observed GNAS imprinting defects. That the mother of case 4 has mild brachydactyly but no loss of exon A/B methylation (due to the paternal inheritance of the 3-kb deletion) supports this possibility. Nevertheless, if the AHO-like features are related to GNAS imprinting defects (possibly for cases 1–3), Gs imprinting may contribute, at least in some patients, to the pathogenesis of AHO. Consistent with recent evidence that obesity is a more prominent feature in PHP-Ia than in PPHP (17), this interpretation implies that Gs expression is subject to allelic bias in a larger number of tissues than currently recognized and that the degree of this allelic bias varies among individuals. Indeed, a previous report has demonstrated modest maternal predominance of Gs expression in one of 19 human bone samples (18).

The cases presented here were from a total of 22 AHO patients of whom 14 showed coding Gs mutations (63%); this is comparable with the mutation identification rate (70%) reported previously for PHP-Ia/PPHP patients by using direct sequence analysis (19, 20). In our study, GNAS methylation defects therefore explain half of the eight cases lacking Gs coding mutations. Furthermore, a maternal deletion involving both exon A/B and the Gs promoter could explain the observed AHO phenotype and loss of methylation in case 1 because she was homozygous for polymorphisms throughout the region (data not shown). However, a Southern analysis using a probe close to the exon A/B region revealed wild-type hybridizing bands only (data not shown). Moreover, an analysis of gene dosage using real-time PCR argued against a loss of exon A/B copy number in case 1 (data not shown). These findings are consistent with the presence of both parental alleles but cannot rule out a large deletion in this region.

In summary, we have revealed a subpopulation of PHP patients who coexhibit mild AHO and GNAS imprinting defects. We thus propose that in patients with hormone resistance and AHO-like features, both mutational analysis of Gs-coding GNAS exons and evaluation of GNAS imprinting should be considered.

	Footnotes

 
This work was partially supported by Grant RCMN (C03/08) from the Instituto de Salud Carlos III, Madrid, Spain; a grant from Pfizer Laboratories; grants from the National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland (RO1 46718-10 to H.J. and KO1 DK-062973 to M.B.); and a grant from the Bundesministerium für Bildung und Forschung (German Ministry for Research and Education; GFGM01141901 to W.A. and O.H.). G.P.d.N. and J.R.B. are FIS Research Scientists supported by the Spanish Ministry of Health (Fellowship 03/0064 and 99/3076, respectively).

Disclosure Statement: The authors have nothing to disclose.

First Published Online April 3, 2007

Abbreviations: AHO, Albright’s hereditary osteodystrophy; CT, computed tomography; fT4, free T₄; PHP, pseudohypoparathyroidism; PPHP, pseudopseudohypoparathyroidism.

Received October 19, 2006.

Accepted March 23, 2007.

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