Jansen's metaphyseal chondrodysplasia (JMC) is a rare disorder caused by activating mutations in the parathyroid hormone (PTH)/PTH-related peptide (PTHrP) receptor (PTH1R). Patients exhibit short stature, dysmorphic bones, and severe growth plate abnormalities, as well as hypercalcemia, hypercalciuria, hypophosphatemia, and reduced plasma PTH levels. Humanized PTH1R (hPTH1R) mice expressing the H223R-hPTH1R JMC mutation die early without breeding. We therefore generated and characterized a stable mouse line expressing the T410R-hPTH1R allele, which confers a milder disease phenotype in patients. Mutant mice show near-normal longevity and reproductive capacity yet exhibit a profound skeletal phenotype characteristic of the disease. The long bones of T410R mice are markedly misshapen and have expanded metaphyses with disarrayed chondrocyte zones in growth plates and reduced primary spongiosa. PET/CT scanning revealed diminished uptake of [18F]-sodium fluoride in the growth plate area, consistent with reduced mineralization and vascularization. Genetic ablation of Hdac4 rescued the growth plate abnormalities in T410R mice, thereby establishing the PTH1R-Gαs-cAMP-PKA-SIK3-HDAC4/5 pathway as the main mediator of growth plate abnormalities in JMC. Serum calcium was elevated and endogenous PTH was suppressed in T410R mice, and both parameters could be normalized by acute injection of an optimized PTH inverse agonist peptide. The T410R mouse thus represents a stable animal model of JMC that recapitulates the abnormalities in skeletal development and mineral ion homeostasis which characterize this disease. The mice should help efforts to further define the cellular and molecular mechanisms underlying the JMC phenotype and to develop a potential mode of therapy.

Abstract Disclosure: J. Höppner: None. P. Hanna: None. M.N. Wein: None. H. Jueppner: None. T.J. Gardella: None. R. Civitelli: None. I.A. Portales-Castillo: None. Abstract The parathyroid hormone receptor-1 (PTH1R) plays a key role in bone development and acts in the growth plates in response to PTHrP ligand. Eiken syndrome is characterized by a delay in bone mineralization and is caused by homozygous PTH1R mutations. One such mutation, R485X, truncates the receptor's C-tail and removes serine phosphorylation sites involved in βarrestin binding. In HEK293 cells, R485X-hPTH1R exhibits deficient interaction with βarrestin and increased cAMP signaling both basally and in response to PTHrP (Portales-Castillo et al., Comms. Biology 2023). To understand how the R485X mutation causes delayed bone mineralization, we generated humanized R485X-hPTH1R knock-in mice. Homozygous hPTH1RR485X/R485X mice closely recapitulate the delayed bone mineralization seen in Eiken patients, as skeletal whole mount preparations from neonatal mutant mice showed reduced Alizarin red staining compared to WT controls, and metatarsal explants from the mutant mice showed a pronounced absence of mineralized bone. Tails of hPTH1RR485X/R485X mice were ∼50% shorter than those of WT littermates, and H&E-stained sections revealed only proliferative chondrocytes in the growth plates of mutant mice. This delay in growth plate chondrocyte maturation in hPTH1RR485X/R485X mice resolved with age, although older long bones and tails remained smaller in size than WT controls (Höppner et al. Presented at ASBMR 2023).Based on our in vitro findings and initial mouse characterization, we reasoned that the phenotype of Eiken mice may be explained by increased PTHrP/PTH1R signaling in growth plate chondrocyte. The class IIa histone deacetylase HDAC4 suppresses chondrocyte maturation downstream of PTH1R signaling. Therefore, we generated compound mutant mice bearing both Hdac4 deletion and the mutant R485X PTH1R allele. Indeed, Hdac4 deletion largely rescued the mineralization defect apparent in metatarsals of P1 hPTH1RR485X/R485X mice. We then assessed the contribution of endogenous PTHrP to the Eiken-like phenotype by generating hPTH1RR485X/R485X/PTHrPflox/+/Col2-Cre(tg) mice. Remarkably, these mice exhibit approximately the same tail lengths as hPTH1R-WT littermate controls, indicating that reduced PTHrP production can rescue the effects of homozygosity for R485X-PTH1R. In line, vertebral growth plates of the rescued mice exhibited normal zones of chondrocytes, including hypertrophic cells. The overall results support a disease mechanism for Eiken syndrome by which excess cAMP signaling by PTH1R-R485X, as induced in part by endogenous PTHrP, results in delayed chondrocyte differentiation and bone mineralization. Further studies employing βarr1/2-KO mice may help shed light on the specific roles of βarrestins in growth plate development by PTH1R in Eiken syndrome. Presentation: 6/3/2024

ABSTRACT Pseudohypoparathyroidism type Ib (PHP1B) is characterized predominantly by resistance to parathyroid hormone (PTH) leading to hypocalcemia and hyperphosphatemia. These laboratory abnormalities are caused by maternal loss-of-methylation (LOM) at GNAS exon A/B, which reduces in cis expression of the stimulatory G protein α-subunit (Gsα). Paternal Gsα expression in proximal renal tubules is silenced through unknown mechanisms, hence LOM at exon A/B reduces further Gsα protein in this kidney portion, leading to PTH resistance. In a previously reported PHP1B family, affected members showed variable LOM at exon A/B, yet no genetic defect was found by whole-genome sequencing despite linkage to GNAS. Using targeted long-read sequencing (T-LRS), we discovered an approximately 2800-bp maternally inherited retrotransposon insertion nearly 1200 bp downstream of exon XL not found in public databases or in 13,675 DNA samples analyzed by short-read whole-genome sequencing. T-LRS data furthermore confirmed normal methylation at exons XL, AS, and NESP and showed that LOM comprising exon A/B is broader than previously thought. The retrotransposon most likely causes the observed epigenetic defect by impairing function of a maternally derived NESP transcript, consistent with findings in mice lacking full-length NESP mRNA and in PHP1B patients with deletion of exon NESP and adjacent intronic sequences. In addition to demonstrating that T-LRS is an effective strategy for identifying a small disease-causing variant that abolishes or severely reduces exon A/B methylation, our data demonstrate that this sequencing technology has major advantages for simultaneously identifying structural defects and altered methylation. © 2022 American Society for Bone and Mineral Research (ASBMR).

Like other secreted peptides, nascent parathyroid hormone (PTH) is synthesized with a pre- and a pro-sequence (25 and 6 amino acids, respectively). These precursor segments are sequentially removed in parathyroid cells before packaging into secretory granules. Three patients from two unrelated families who presented during infancy with symptomatic hypocalcemia were found to have a homozygous serine (S) to proline (P) change affecting the first amino acid of the mature PTH. Unexpectedly, biological activity of synthetic [P1]PTH(1-34) was indistinguishable from that of unmodified [S1]PTH(1-34). However, in contrast to conditioned medium from COS-7 cells expressing prepro[S1]PTH(1-84), medium from cells expressing prepro[P1]PTH(1-84) failed to stimulate cAMP production despite similar PTH levels when measured by an intact assay that detects PTH(1-84) and large amino-terminally truncated fragments thereof. Analysis of the secreted, but inactive PTH variant led to the identification of pro[P1]PTH(−6 to +84). Synthetic pro[P1]PTH(−6 to +34) and pro[S1]PTH(−6 to +34) had much less bioactivity than the corresponding PTH(1-34) analogs. Unlike pro[S1]PTH(−6 to +34), pro[P1]PTH(−6 to +34) was resistant to cleavage by furin suggesting that the amino acid variant impairs preproPTH processing. Consistent with this conclusion, plasma of patients with the homozygous P1 mutation had elevated proPTH levels, as determined with an in-house assay specific for pro[P1]PTH(−6 to +84). In fact, a large fraction of PTH detected by the commercial intact assay represented the secreted pro[P1]PTH. In contrast, two commercial biointact assays that use antibodies directed against the first few amino acid residues of PTH(1-84) for capture or detection failed to detect pro[P1]PTH.

Abstract Activating parathyroid hormone (PTH)/PTH‐related Peptide (PTHrP) receptor (PTH1R) mutations causes Jansen's metaphyseal chondrodysplasia (JMC), a rare disease characterized by growth plate abnormalities, short stature, and PTH‐independent hypercalcemia. Previously generated transgenic JMC mouse models, in which the human PTH1R allele with the H223R mutation (H223R‐PTH1R) is expressed in osteoblasts via type Ia1 collagen or DMP1 promoters cause excess bone mass, while expression of the mutant allele via the type IIa1 collagen promoter results in only minor growth plate changes. Thus, neither transgenic JMC model adequately recapitulates the human disease. We therefore generated “humanized” JMC mice in which the H223R‐PTH1R allele was expressed via the endogenous mouse Pth1r promoter and, thus, in all relevant target tissues. Founders with the H223R allele typically died within 2 months without reproducing; several mosaic male founders, however, lived longer and produced F1 H223R‐PTH1R offspring, which were small and exhibited marked growth plate abnormalities. Serum calcium and phosphate levels of the mutant mice were not different from wild‐type littermates, but serum PTH and P1NP were reduced significantly, while CTX‐1 and CTX‐2 were slightly increased. Histological and RNAscope analyses of the mutant tibial growth plates revealed markedly expanded zones of type II collagen‐positive, proliferating/prehypertrophic chondrocytes, abundant apoptotic cells in the growth plate center and a progressive reduction of type X collagen‐positive hypertrophic chondrocytes and primary spongiosa. The “humanized” H223R‐PTH1R mice are likely to provide a more suitable model for defining the JMC phenotype and for assessing potential treatment options for this debilitating disease of skeletal development and mineral ion homeostasis. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Background Maternal inactivating GNAS mutations lead to pseudohypoparathyroidism 1A (PHP1A), newly classified as inactivating parathyroid hormone (PTH)/PTHrP-signaling disorder type 2 of maternal inheritance (iPPSD2). Patients present with resistance to PTH and other hormones, subcutaneous ossifications, brachydactyly, short stature, and early-onset obesity. They can be born small for gestational age (SGA) and may present with growth hormone (GH) deficiency. The use of recombinant human GH (rhGH) therapy has been sporadically reported, yet we lack data on the long-term efficacy and safety of rhGH, as well as on adult height. Objective Our multicenter, retrospective, observational study describes growth in patients treated with rhGH in comparison with untreated iPPSD2/PHP1A controls. Methods We included 190 patients, of whom 26 received rhGH. Height, weight, body mass index at various time points, and adult height were documented. We analyzed the effect of rhGH on adult height by using linear mixed models. Results Adult height was available for 11/26 rhGH-treated individuals and for 69/164 controls. Patients treated with rhGH showed a gain in height of 0.7 standard deviation scores (SDS) after 1 year (CI +0.5 to +0.8, P < .001) and of 1.5 SDS after 3 years (CI +1.0 to +2.0, P < .001). Additionally, there was a clear beneficial impact of rhGH on adult height when compared with untreated controls, with a difference of 1.9 SDS (CI +1.1 to +2.7, P < .001). Body mass index SDS did not vary significantly upon rhGH therapy. Conclusion Recombinant human growth hormone treatment of iPPSD2/PHP1A patients with short stature improves growth and adult height. More studies are needed to confirm long-term efficacy and safety.

Objective Pseudohypoparathyroidism and related disorders belong to a group of heterogeneous rare diseases that share an impaired signaling downstream of Gsα-protein-coupled receptors. Affected patients may present with various combination of symptoms including resistance to PTH and/or to other hormones, ectopic ossifications, brachydactyly type E, early onset obesity, short stature and cognitive difficulties. Several years ago we proposed a novel nomenclature under the term of inactivating PTH/PTHrP signaling disorders (iPPSD). It is now of utmost importance to validate these criteria and/or improve the basis of this new classification. Design Retrospective study of a large international series of 459 probands and 85 relatives molecularly characterized. Methods Information on major and minor criteria associated with iPPSD and genetic results were retrieved from patient files. We compared the presence of each criteria according to the iPPSD subtype, age and gender of the patients. Results More than 98% of the probands met the proposed criteria for iPPSD classification. Noteworthy, most patients (85%) presented a combination of symptoms rather than a single sign suggestive of iPPSD and the overlap among the different genetic forms of iPPSD was confirmed. The clinical and molecular characterization of relatives identified familial history as an additional important criterion predictive of the disease. Conclusions The phenotypic analysis of this large cohort confirmed the utility of the major and minor criteria and their combination to diagnose iPPSD. This report shows the importance of having simple and easily recognizable signs to diagnose with confidence these rare disorders and supports a better management of patients.

CONTEXT:Maternally inherited STX16 deletions that cause loss of methylation at GNAS exon A/B and thereby reduce Gsα expression are the most frequent cause of autosomal dominant pseudohypoparathyroidism type Ib (AD-PHP1B). Early identification of these disease-causing variants in the children of affected and unaffected female carriers would prompt treatment with calcium and calcitriol once parathyroid hormone (PTH) levels increase, thereby preventing hypocalcemia and associated complications.OBJECTIVE:This study aimed to determine when PTH and calcium abnormalities develop after birth if a STX16 deletion is inherited maternally.METHODS:Forty-four children of affected (n = 7) or unaffected (n = 7) females with a STX16 deletion were investigated for the presence of these variants. If a deletion was identified, measurement of PTH, calcium, phosphate, and thyrotropin (TSH) was advised.RESULTS:The STX16 deletion that causes AD-PHP1B was identified in 25 children. Pretreatment laboratory results were available for 19 of those cases. Elevated PTH levels were detected by 2 years of age, and these were progressively higher if laboratory testing was first performed after establishing the genetic defect later in life. Total serum calcium levels remained within normal limits until about 5 years of age. TSH levels showed no consistent rise over time.CONCLUSION:Establishing whether a STX16 deletion is inherited from a female carrier of a disease-causing variant rapidly establishes the diagnosis of AD-PHP1B. Several years before overt hypocalcemia developed, PTH levels increased, thereby establishing the onset of PTH resistance. Our findings provide diagnostic guidance and when treatment with calcium and calcitriol should be considered in order to prevent hypocalcemia and associated sequelae.

Context: Pseudohypoparathyroidism type Ib (PHP1B) is characterized by hypocalcemia and hyperphosphatemia due to parathyroid hormone resistance in the proximal renal tubules. Maternal pathogenic STX16/GNAS variants leading to maternal epigenetic GNAS changes impair expression of the stimulatory G protein alpha-subunit (Gsa) thereby causing autosomal dominant PHP1B. In contrast, genetic defects responsible for sporadic PHP1B (sporPHP1B) remain mostly unknown. Objective: Determine whether PHP1B encountered after in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) causes GNAS remethylation defects similar to those in sporPHP1B. Design: Retrospective analysis. Results: Nine among 36 sporPHP1B patients investigated since 2000, all with loss of methylation (LOM) at the 3 maternal GNAS differentially methylated regions (DMRs) and gain of methylation at the paternal NESP DMR, had been conceived through IVF or ICSI. Besides abnormal GNAS methylation, IVF/ICSI PHP1B cases revealed no additional imprinting defects. Three of these PHP1B patients have dizygotic twins, and 4 have IVF/ICSI-conceived siblings, all with normal GNAS methylation; 2 unaffected younger siblings were conceived naturally. Conclusion: Sporadic and IVF/ICSI-conceived PHP1B patients revealed indistinguishable epigenetic changes at all 4 GNAS DMRs, thus suggesting a similar underlying disease mechanism. Given that remethylation at the 3 maternal DMRs occurs during oogenesis, male factors are unlikely to cause LOM postfertilization. Instead, at least some of the sporPHP1B variants could be caused by a defect or defects in an oocyte-expressed gene that is required for fertility and for re-establishing maternal GNAS methylation imprints. It remains uncertain, however, whether the lack of GNAS remethylation alone and the resulting reduction in Gsa expression is sufficient to impair oocyte maturation.

CONTEXT Pseudohypoparathyroidism type 1B (PHP1B), also referred to as inactivating PTH/PTHrP Signaling Disorder (iPPSD), is characterized by proximal renal tubular resistance to parathyroid hormone (PTH) leading to hypocalcemia, hyperphosphatemia and elevated PTH values. Autosomal dominant PHP1B (AD-PHP1B) with loss-of-methylation at the maternal GNAS A/B:TSS-DMR (transcription start site-differentially methylated region) alone can be caused by maternal deletions involving STX16. OBJECTIVES Characterize a previously not reported AD-PHP1B family with loss-of-methylation at GNAS A/B:TSS-DMR, but without evidence for a STX16 deletion on the maternal allele and assess GNAS-AS2:TSS-DMR methylation. PATIENTS AND METHODS DNAs from 24 patients and 10 controls were investigated. AD-PHP1B patients without STX16 deletion from a single family (n=3), AD-PHP1B patients with STX16 deletion (n=9), sporPHP1B (n=10), unaffected controls (n=10), patUPD20 (n=1), and matUPD20 (n=1). Methylation and copy number analyses were performed by pyrosequencing, MS-MPLA, and MLPA, respectively. RESULTS Molecular cloning of PCR-amplified, bisulfite-treated genomic DNA from healthy controls revealed evidence for two distinct GNAS-AS2:TSS-DMR subdomains, named AS2-1 and AS2-2, which showed 16.0±2.3% and 31.0±2.2% methylation, respectively. DNA from affected members of a previously not reported AD-PHP1B family without the known genetic defects revealed incomplete LOM (loss-of-methylation) at GNAS A/B:TSS-DMR, normal methylation at the three well-established maternal and paternal DMRs, and, surprisingly, increased methylation at AS2-1 (32.9±3.5%), but not at AS2-2 (30.5±2.9%). CONCLUSION The distinct methylation changes at the novel GNAS-AS2:TSS-DMR will help characterize further different PHP1B/iPPSD3 variants and will guide the search for underlying genetic defects, which may provide novel insights into the mechanisms underlying GNAS methylation.

Patients affected by pseudohypoparathyroidism (PHP) or related disorders are characterized by physical findings that may include brachydactyly, a short stature, a stocky build, early-onset obesity, ectopic ossifications, and neurodevelopmental deficits, as well as hormonal resistance most prominently to parathyroid hormone (PTH). In addition to these alterations, patients may develop other hormonal resistances, leading to overt or subclinical hypothyroidism, hypogonadism and growth hormone (GH) deficiency, impaired growth without measurable evidence for hormonal abnormalities, type 2 diabetes, and skeletal issues with potentially severe limitation of mobility. PHP and related disorders are primarily clinical diagnoses. Given the variability of the clinical, radiological, and biochemical presentation, establishment of the molecular diagnosis is of critical importance for patients. It facilitates management, including prevention of complications, screening and treatment of endocrine deficits, supportive measures, and appropriate genetic counselling. Based on the first international consensus statement for these disorders, this article provides an updated and ready-to-use tool to help physicians and patients outlining relevant interventions and their timing. A life-long coordinated and multidisciplinary approach is recommended, starting as far as possible in early infancy and continuing throughout adulthood with an appropriate and timely transition from pediatric to adult care.

Objectives The objective of this paper is to report a peculiar case of a patient with pseudohypoparathyroidism type 1b (PHP1B). Pseudohypoparathyroidism (PHP) refers to a group of disorders characterized by hypocalcemia, hyperphosphatemia, and elevated parathyroid hormone (PTH) concentrations as the result of end-organ unresponsiveness to PTH. Case presentation We present a 14-year-old boy, who was admitted with severe symptomatic hypocalcaemia, absence of dysmorphic features and Albright's hereditary osteodystrophy features. Laboratory investigations revealed markedly low serum calcium, high phosphate, markedly elevated PTH levels and vitamin D insufficiency, while magnesium, albumin, ALP and TSH were normal. The clinical and laboratory findings were consistent with PHP1B. Molecular analysis revealed loss of methylation at the AB DMR of the GNAS locus, confirming the diagnosis. Yet no STX16 deletion was detected. Conclusions It is possible that delSTX16- patients carry a defect in an element that controls the methylation both at the GNAS-A/B DMR and at the GNAS-AS2. This rare case emphasizes the need of individualized molecular analysis in PHP1B patients in order to elucidate the possible molecular defect.