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Genetic Analysis and Evaluation of Resistance to Thyrotropin and Growth Hormone-Releasing Hormone in Pseudohypoparathyroidism Type Ib

Department of Medical Sciences (G.M., S.B., A.G.L., P.B.-P., A.S.), Endocrine Unit, Fondazione Policlinico Instituto di Ricovero e Cura a Carattere Scientifico, University of Milan, 20122 Milan, Italy; Pediatric Endocrinology and Institut National de la Santé et de la Recherche Médicale U561 (A.L.), St. Vincent de Paul Hospital, 75014 Paris, France; Department of Pediatrics (M.M.), Istituto Giannina Gaslini Instituto di Ricovero e Cura a Carattere Scientifico, University of Genova, 16145 Genova, Italy; Department of Pediatrics (M.C.), Fondazione Instituto di Ricovero e Cura a Carattere Scientifico Policlinico S. Matteo, University of Pavia, 27100 Pavia, Italy; and Endocrinology and Diabetology Unit (S.C.), Department of Medical-Surgical Sciences, Policlinico San Donato Instituto di Ricovero e Cura a Carattere Scientifico, San Donato Milanese, University of Milan, Milan, 20095 Italy

Address all correspondence and requests for reprints to: Giovanna Mantovani, M.D., Ph.D., Unità di Endocrinologia, Pad. Granelli, Fondazione Ospedale Maggiore Instituto di Ricovero e Cura a Carattere Scientifico, Via Francesco Sforza, 35, 20122 Milano, Italy. E-mail: giovanna.mantovani{at}unimi.it.

	Abstract

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Context: Pseudohypoparathyroidism (PHP) types Ia and Ib, are caused by mutations in GNAS exons 1–13 and GNAS methylation defects, respectively. PHP-Ia patients show Albright hereditary osteodystrophy (AHO) and resistance toward PTH and additional hormones, whereas PHP-Ib patients do not have AHO and hormone resistance is limited to PTH and, as reported in one paper, TSH. No study addressed the question of GH deficiency in PHP-Ib patients.

Objectives: The objective of the study was to screen patients with clinically diagnosed PHP-Ib for genetic defects and investigate the presence of resistance to TSH and GHRH.

Patients/Methods: We investigated GNAS differential methylation and STX16 microdeletions in genomic DNA from 10 patients with clinical diagnosis of sporadic PHP-Ib, i.e. PTH resistance without AHO. Resistance to GHRH was assessed by GH response to GHRH plus arginine. Thyroid function and ultrasonography were also evaluated.

Results: Molecular analysis showed GNAS cluster imprinting defects in all PHP-Ib patients and the first de novo STX16 deletion in one apparently sporadic patient. Subclinical or clinical hypothyroidism due to resistance to TSH was present in nine of 10 patients, whereas a preserved GH response to a GHRH plus arginine test was present in all patients, with one exception.

Conclusions: We report the first molecular analysis of Italian patients with PHP-Ib. Clinical investigation shows that, like PHP-Ia patients, PHP-Ib patients are resistant to TSH, whereas they maintain a normal responsiveness to GHRH, at variance with PHP-Ia patients. These data provide new information on this rare disease and emphasize the clinical heterogeneity of genetic defects within GNAS.

	Introduction

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PSEUDOHYPOPARATHYROIDISM (PHP) refers to a heterogeneous group of rare metabolic disorders characterized by hypocalcemia and hyperphosphatemia due to PTH resistance (1, 2). The two main subtypes of PHP, PHP types Ia and Ib, are caused by genetic alterations within or upstream of the GNAS locus. GNAS gives rise to several different transcripts, including Gs, XLs (extra-large variant of Gs), neuroendocrine secretory protein (NESP) 55, and several additional sense and antisense transcripts. The complexity of the GNAS locus is furthermore reflected by the parent-specific methylation pattern of most of its different promoters (Fig. 1A) (reviewed in Ref. 3).

View larger version (25K): [in this window] [in a new window]   FIG. 1. A, General organization and imprinting of GNAS. The maternal and paternal alleles of GNAS are shown. Methylated regions (Meth) are indicated and active promoters are represented by arrows in the direction of transcription. The alternative first exons and common exon 2 are shown as rectangles with exon 1 being the first coding exon for Gs. Common downstream exons 3–13 as well as the downstream exons of the antisense transcript (AS) are not shown. Exon 1 is paternally imprinted in some tissues, indicated by broken arrow. The diagram is not drawn to scale. B, Mean GH response to GHRH plus arginine injection in the 10 PHP-Ib patients included in the study. The panel shows the mean GH peak in our series of adult PHP-Ia (6 ) and PHP-Ib patients (present series) as well as in an in-house age- and sex-matched control population. *, P < 0.01 vs. control. C, Methylation analysis of GNAS DMRs. Genomic DNA from each subject included in the study was bisulfite treated, and each GNAS DMR was amplified using specific primers. PCR products were digested using methylation-sensitive restriction enzymes (for details see supplemental Table 1). Wild-type A/B, XL, and AS DMRs are digested by the enzyme used into two bands, one corresponding to the unmethylated paternal allele (uncut) and one to the methylated maternal allele (cut); the absence of the digested band indicates loss of methylation on the maternal allele. In the case of NESP DMR, the epigenetic defect is represented by a gain of methylation on the maternal allele, indicated by the completely cut PCR product (some undigested DNA is often still visible, but the digested band is always brighter); wild-type NESP methylation status is indicated by the presence of two bands. D, STX16 deletion analysis performed by long-range and multiplex PCR. Long-range PCR analysis is performed using only primers A and B (upper panel). In this case, normal samples show a 4.5-kb PCR band, but more often no band can be amplified; subjects affected by the deletion show a 1387-bp PCR fragment that can be amplified only on the deleted allele. When multiplex PCR is used for the identification of the deletion (lower panel), normal subjects show two PCR products (1496 and 966 bp, the 4.5-kb fragment is not usually amplified), whereas deleted samples show an additional 1387-bp band amplified on the deleted allele (for primers position, see supplemental Fig. 1). We show the results obtained for a group of representative samples: patient 1 (P1), patient 2 (P2), patient 3 (P3), patient 7 (P7), patient 7’s mother (M7), and a normal subject as control (C).

In this study we first analyzed the presence of GNAS imprinting defects in a series of Italian patients with the diagnosis of PHP-Ib based on clinical criteria, and we then investigated the presence of resistance to the action of TSH and GHRH.

	Subjects and Methods

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Methylation analysis of GNAS

Genomic DNA was isolated from patients’ peripheral blood samples (Nucleon BACC2; Amersham Life Science Europe, Milan, Italy). Differential methylation of GNAS differentially methylated regions (DMRs) A/B, AS, XL, and NESP was assessed by restriction enzymatic analysis of PCR products amplified from bisulfite-treated genomic DNA, as previously reported (13). The supplemental Table 1 (published as supplemental data on The Endocrine Society’s Journals Online Web site at http://jcem.endojournals.org) shows the sequence of the primers and the restriction enzymes used for each GNAS DMR.

Detection of STX16 microdeletions

We investigated the presence of STX16 3-Kb microdeletion in patients’ genomic DNA by both long-range and multiplex PCR techniques, as previously described (14). Briefly, long-range PCR was performed using Platinum Taq DNA polymerase (Invitrogen S.R.L., Milan, Italy) according to the manufacturer’s conditions and a pair of primers located at each boundary of the 3-kb deletion that give rise to a 4.5-kb fragment on the wild-type allele and a smaller 1.387-kb fragment in case of a 3-kb deleted STX16 allele. For multiplex PCR we simultaneously used two pairs of primers giving rise to PCR products of different length, depending on the presence of the deleted allele (supplemental Fig. 1, published as supplemental data on The Endocrine Society’s Journals Online Web site at http://jcem.endojournals.org).

Testing

All subjects were tested with GHRH [GHRH 1–29; Geref, Serono, Italy; 1 µg/kg body weight iv at time 0] plus arginine (0.5 g/kg body weight, L-arginine monohydrochloride iv, from time 0 over 30 min) after overnight fasting, as previously reported (6). Blood samples were drawn from a indwelling catheter inserted in an antecubital vein at –15 min and at times 0, 15, 30, 45, 60, and 90 min. Normal GH peaks after stimulation in normal-weight subjects were greater than 16.5 µg/liter; partial GH deficiency, 9–16.5 µg/liter; and complete GH deficiency, less than 9 µg/liter (15). Serum GH levels were measured by an immunofluorimetric assay method supplied by AutoDelfia kit (Wallac OY, Finland). Serum IGF-I was also measured in all patients (Mediagnost, Tubingen, Germany).

Informed consent was obtained from all subjects involved in the study. The study was approved by the local ethical committee.

	Results

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Patients

The study included 10 patients (four females and six males, aged 16–48 yr at the time of GH secretion evaluation) in whom diagnosis of PHP-Ib was based on the lack of AHO manifestations together with PTH resistance (hypocalcemia with raised serum PTH levels). The age at diagnosis ranged between 2 and 48 yr. All patients showed resistance to TSH, as documented by raised serum TSH levels, in the absence of antithyroid antibodies and the presence of a normal thyroid ultrasonography (data not shown). Moreover, patient 4 also showed modestly low free T₄ levels. There was no evidence of other endocrine defects in these patients. In particular, the onset of puberty was regular and so was the menstrual cycle in females. Gonadotropin, ACTH and cortisol levels as well as estradiol levels in females and testosterone levels in males were in the normal range in all patients (data not shown). Each patient’s final stature was in accordance with the corresponding target height, and all subjects had a normal body mass index. The required therapy (calcium, vitamin D, and L-thyroxine) was not discontinued during the study. Clinical and biochemical details are shown in Table 1.

The mean GH response after GHRH plus arginine was 38.47 ± 18.9 µg/liter (range 12.3–72.4). In particular, nine patients had a normal GH peak greater than 16.5 µg/liter during the provocative test and basal IGF-I levels in the normal range according to age and sex. Conversely, patient 9 had borderline/low GH peak and modestly low IGF-I levels (Table 1 and Fig. 1B). This 48-yr-old patient presented with extremely low calcium levels (4.3 mg/dl), a history of seizures since infancy, and evidence of extended intracranial calcifications at computed tomography scan. Additional stimulation tests exploring the pituitary function excluded the presence of other deficiencies.

Genetic analysis

The 10 patients included in the study showed methylation defects at GNAS DMRs, thus confirming the diagnosis of PHP-Ib at molecular level (Table 1 and Fig. 1C). In particular, all patients showed loss of methylation at exon A/B, a condition sufficient to determine PHP-Ib manifestations. Nine patients were true sporadic PHP-1b cases, also showing loss of methylation at XL and AS DMRs, and gain of methylation at NESP DMR (Table 1 and Fig. 1C). As described in sporadic PHP-1b, these patients did not show STX16 microdeletions (Fig. 1D). Conversely, patient 7 presented a 3-kb deletion in STX16 gene and methylation defect only at A/B DMR, despite the lack of family history of PHP-Ib (Fig. 1, C and D). Because the mode of inheritance of PHP-1b is maternal, we analyzed patient 7’s mother’s DNA (M7 in Fig. 1D), which resulted normal for both GNAS epigenetic analysis and the STX16 deletion.

	Discussion

Top Abstract Introduction Subjects and Methods Results Discussion References

 
Here we report the screening for methylation defects at the GNAS locus in a series of patients with a diagnosis of PHP-Ib based on clinical criteria and provide new information on the clinical characteristics of this rare disease. This study, which represents the first Italian series of patients with PHP-Ib and one of the largest series in the literature with complete clinic and genetic assessment, included 10 patients who displayed the typical features of PHP-Ib, i.e. resistance to PTH in the absence of AHO. After the first report in 2003 of borderline to mild TSH resistance in half of PHP-Ib patients (8), this study first confirms and extends this observation. Indeed, nearly all patients of this series had raised serum TSH levels in the absence of antithyroid antibodies and the presence of a normal thyroid ultrasonography. As observed in PHP-Ia, PHP-Ib patients also had subclinical hypothyroidism, with the exception of one patient who developed a mild hypothyroidism. Conversely, in contrast to what occurs in PHP-Ia, no PHP-Ib patient showed resistance to gonadotropins.

Molecular analysis demonstrated methylation defects at the GNAS locus in all patients, confirming the clinical diagnosis. It is known that there are two variants of PHP-Ib that differ at the molecular level, i.e. a familial autosomal dominant form (AD-PHP-Ib) and a sporadic form. In the very few series of PHP-Ib patients reported in the literature, most individuals with AD-PHP-Ib have a maternally inherited microdeletion within STX16, a gene thought to contain an imprinting control element required for the establishment and/or maintenance of normal methylation at the GNAS A/B exon. Accordingly, these patients, in addition to STX16 deletion, show loss of methylation at exon A/B, thus leading to Gs transcription silencing in the proximal renal tubules. Conversely, individuals with sporadic PHP-Ib display broader methylation defects at the GNAS locus, in the absence of microdeletions within STX16 (16). Our series included 10 patients with apparently sporadic PHP-Ib. All of them showed loss of methylation at exon A/B, a condition sufficient to determine the clinical manifestations of PHP-Ib (9, 13, 14, 16). In addition, nine patients revealed classical epigenetic alterations involving different GNAS DMRs, with no STX16 deletion (Table 1). Unexpectedly, in patient 7 we detected the 3-kb STX16 deletion classically reported for familial cases. Because the mother’s DNA was normal, this is the first report of a de novo STX16 deletion with consequent A/B loss of methylation, this finding being important for genetic counseling.

It has become clear that PHP-Ia and PHP-Ib share many genetic similarities because both diseases end up with silencing or reduction of Gs transcription in selected tissues. It is therefore conceivable that these two disorders may share more endocrine defects than those originally identified. The presence here of confirmed TSH resistance in PHP-Ib patients strongly supports this hypothesis. Thus, we decided to screen PHP-Ib patients for GH deficiency, the latest endocrine defect identified in PHP-Ia. To evaluate the presence of resistance to GHRH, patients were subjected to a combined provocative test that explores the maximal secretory response to GHRH in association with the arginine-induced inhibition of somatostatin release (15, 17). This test, which is one of the most reliable tests to distinguish normal subjects from GH-deficient patients during the whole life span (15, 17, 18), showed a preserved responsiveness to GHRH in all patients, as expected by the normal stature/puberty in our probands, with the exception of patient 9, who showed partial GH deficiency and slightly reduced IGF-I levels. Because this patient had a history of long-standing and severe hypocalcemia, seizures since infancy, and extended intracranial calcifications at computed tomography scan, it is tempting to speculate that impaired GH secretion in this patient might be secondary to these events.

Taken together these data indicate that, unlike what happens for Gs mutations in PHP-Ia, disruption of physiological GNAS imprinting status does not affect Gs transcription in somatotrophs. This is in accordance with the hypothesis that GNAS methylation defects lead to Gs silencing with consequent hormone resistance only in selected tissues expressing a tissue-specific factor that binds to a site within exon A/B DMR and suppresses the Gs promoter only on the unmethylated paternal allele (16).

Finally, the observation that PHP-Ia patients generally develop hypocalcemia, hypothyroidism, and hypogonadism over childhood supports the hypothesis that imprinting can be a process evolving in postnatal life, as also suggested by the biallelic expression of the Gs gene in different human fetal tissues (19). All the PHP-Ib patients considered in this study have been investigated for GH deficiency at adulthood, and this should exclude that mild forms of GH deficiency might develop later on in these patients.

In conclusion, we report the first molecular analysis of Italian patients with PHP-Ib and the first case of a de novo STX16 deletion in an apparently sporadic PHP-Ib patient. Clinical evaluation showed that, in analogy with PHP-Ia patients, almost all PHP-Ib patients were resistant to TSH action. Conversely, in contrast with what is observed in PHP-Ia, patients with PHP-Ib displayed normal responsiveness to GHRH and normal IGF-I levels, pointing out the clinical heterogeneity of genetic defects within the GNAS locus.

	Footnotes

 
This work was partially supported by Associazione Italiana Ricerca Cancro, Milan, and Ricerca Corrente Funds of Fondazione Policlinico Instituto di Ricovero e Cura a Carattere Scientifico, Milan.

The authors have nothing to disclose.

First Published Online June 26, 2007

¹ G.M. and S.B. contributed equally to this work and should be therefore considered as first coauthors.

Abbreviations: AD, Autosomal dominant form; AHO, Albright hereditary osteodystrophy; DMR, differentially methylated region; NESP, neuroendocrine secretory protein; PHP, pseudohypoparathyroidism.

Received April 17, 2007.

Accepted June 14, 2007.

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