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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.

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.

Case commentary on Foster v Jessen [2021] NIQB 56.

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.

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.

This article compares defamation law in England and Wales with that of Northern Ireland and analyses whether the current law in Northern Ireland is having a ‘chilling effect’ on free speech. At the time of writing, the Northern Ireland Assembly is formally considering adopting legislation based on the Defamation Act 2013 which reformed the law in England and Wales. The article aims to contribute to that debate in Northern Ireland, but it should also be of broader interest as an analysis of the effectiveness of the Defamation Act 2013. The article focuses on three key areas of reform, in both the Defamation Act 2013 and the Northern Ireland Defamation Bill: the presumption of jury trial, the threshold of seriousness, and the public interest defence. It demonstrates that the different approach of the law in Northern Ireland in these areas did not simply occur with the enactment of the 2013 Act, but rather that it started several years before that with a divergence from developments in the common law in England and Wales. The article argues that the difference has been entrenched by the changes in the 2013 Act, and that, in relation to each of those areas, the law in Northern Ireland is now on a singular course and one that can be seen to have a definite ‘chilling effect’ on free speech.

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 (Gsα) 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 Gsα expression is sufficient to impair oocyte maturation.

Background/Aims Individuals on immunosuppressive therapies were among those identified by the UK Department of Health and Chief Medical Officer as clinically extremely vulnerable to COVID-19. Advice on shielding from infection was disseminated by NHS England, primary care and NHS hospital trusts to those identified at highest risk. The British Society for Rheumatology (BSR) developed further risk stratification guidance specifically focused on autoimmune rheumatic disease. As the UK entered a period of nationwide lockdown on 23rd March 2020, the clinically highly vulnerable group were asked to shield from infection by staying at home and avoiding any face-to-face contact for an initial period of 12 weeks. The implications of social isolation, disruption to planned medical care and economic consequences became increasingly recognised. This work aimed to understand the experience and wellbeing of this patient group during week 5 -6 of UK Lockdown, as a guide to how we might best adapt services and address the needs of this group. Methods Short semi-structured telephone interviews were conducted with 141 patients during week five and six of UK lockdown, between 20th April and 1st May 2020. Participants were sampled systematically from the departmental biologic therapy database at Wexham Park Hospital, a general hospital providing services to a diverse population of approximately 450,000 people in Berkshire and South Buckinghamshire. Results Telephone interview was conducted with 141 patients prescribed biologic therapy. Written advice on risk and shielding was received by over 90%. Sixty four percent of respondents were female with a median age of 56 years. Thirty-nine percent of those interviewed fell within highest BSR risk category for whom full shielding was highly recommended, yet at the time of interview 51.1% of respondents reported they had committed to full shielding. Four percent of respondents self-identified as key workers and had continued their usual commitments. Amongst those interviewed, 35% reported significant adverse impact on their emotional and mental wellbeing within the first six weeks of UK lockdown. Isolation, uncertainty, limited physical exercise and cancellations to planned appointments were common themes. Twenty-eight percent of respondents felt their disease control had significantly deteriorated during the ongoing pandemic. Due to prevailing anxiety about the impact of immunosuppression on COVID-19 risk, 5% of patients had adjusted their prescribed therapies without the direct supervision or clinical advice. Conclusion This study gives initial insights into the behaviours and concerns of patients with autoimmune and inflammatory disease on biologic therapy during the first wave of COVID-19 in the UK. Wellbeing and disease management have considerably suffered for many individuals. Further understanding patient experience may help guide restructuring of rheumatology services in next phase of the UK pandemic. Disclosure L.M. Carter: None. H. Irvine: None. A. D’Cunha Kamath: None. A. Sahni: None. P. Hanna: None. N. Rahimi: None. K. Bhamra: None.

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. Objective 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. Methods DNA from 24 patients and 10 controls were investigated. AD-PHP1B patients without STX16 deletion from a single family (n = 5), 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, methylation-sensitive multiplex ligation-dependent probe amplification, and multiplex ligation-dependent probe amplification. Results Molecular cloning of polymerase chain reaction–amplified, bisulfite-treated genomic DNA from healthy controls revealed evidence for 2 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 loss of methylation at GNAS A/B:TSS-DMR, normal methylation at the 3 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.

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.

Pseudohypoparathyroidism type 1A (PHP1A), pseudoPHP (PPHP), and PHP type 1B (PHP1B) are caused by maternal and paternal GNAS mutations and abnormal methylation at maternal GNAS promoter(s), respectively. Adult PHP1A patients are reportedly obese and short, whereas most PPHP patients are born small. In addition to parathyroid hormone (PTH) resistance, PHP1A and PHP1B patients may display early-onset obesity. Because early-onset and severe obesity and short stature are daily burdens for PHP1A patients, we aimed at improving knowledge on the contribution of the GNAS transcripts to fetal and postnatal growth and fat storage. Through an international collaboration, we collected growth and weight data from birth until adulthood for 306 PHP1A/PPHP and 220 PHP1B patients. PHP1A/PPHP patients were smaller at birth than healthy controls, especially PPHP (length Z-score: PHP1A -1.1 ± 1.8; PPHP -3.0 ± 1.5). Short stature is observed in 64% and 59% of adult PHP1A and PPHP patients. PHP1B patients displayed early postnatal overgrowth (height Z-score at 1 year: 2.2 ± 1.3 and 1.3 ± 1.5 in autosomal dominant and sporadic PHP1B) followed by a gradual decrease in growth velocity resulting in normal adult height (Z-score for both: -0.4 ± 1.1). Early-onset obesity characterizes GNAS alterations and is associated with significant overweight and obesity in adults (bodey mass index [BMI] Z-score: 1.4 ± 2.6, 2.1 ± 2.0, and 1.4 ± 1.9 in PPHP, PHP1A, and PHP1B, respectively), indicating that reduced Gsα expression is a contributing factor. The growth impairment in PHP1A/PPHP may be due to Gsα haploinsufficiency in the growth plates; the paternal XLαs transcript likely contributes to prenatal growth; for all disease variants, a reduced pubertal growth spurt may be due to accelerated growth plate closure. Consequently, early diagnosis and close follow-up is needed in patients with GNAS defects to screen and intervene in case of early-onset obesity and decreased growth velocity. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research (ASBMR).

This Article uses social systems theory to examine the increased reliance on a distinction between substantive and procedural international law to resolve cases involving a conflict betweenjus cogensand state immunity. This presents the problem of an evolutionary relationship between international law and the complex differentiation of world society. International law is shown to be structurally related to the segmentary differentiation of states that underwrites modern society's functional differentiation. At the same time, it is shown to be structurally related to the increasing formulation of global norms that result from advanced functional differentiation. The Article then turns to examining the substantive/procedural law distinction as a solution to this dual functional reference problem. The distinction is shown to not only maintain the autopoiesis of law under these difficult conditions, but to also secure law's continued functional relevance in globalized society. This functionalist perspective is used to expose differences in the self-description and operation of international law, to point out how law has been blind to its own coding, and to highlight opportunities for programming law to respond in a more constructive manner to the challenges of globalization.

The ease in digital imaging has led to a decrease in image fidelity where illegal reproduction of multimedia information has become difficult to detect. The most challenging problem is to protect image copyright against illegal copies. Therefore a watermark detection process is required to verify the owner of the image. This paper proposes a blind algorithm to extract the watermark. This algorithm is robust against noise and geometric attacks for grayscale images. Robust feature points are detected using the Harris Detector. In the embedding stage, sequences are placed into regions located around feature points. The sequences used are PN, Gold and decimal. In the extraction process, image features are re-allocated using the same detector. Each feature point is used as a center of an N × N region. This region is moved horizontally and vertically within its neighboring pixels. Each move should be registered in a matrix as a correlation value of this region with the initial sequence. This procedure is repeated for all feature points till we find all the watermarked regions. The maximum correlation obtained will determine the center of the watermarked region. The proposed algorithm is robust against a wide variety of tests and is compared to other schemes.

Pseudohypoparathyroidism 1B (PHP1B) or inactivating PTH/PTHrp Signaling Disorder type 3 (iPPSD3), according to the new classification, presents mainly with hypocalcemia and hyperphosphatemia due to renal resistance towards parathyroid hormone. In addition, patients display early onset obesity and impaired pubertal growth. The disease is due to a methylation defect at one or several Differentially Methylated Regions (DMR) of the GNAS locus, subjected to parental imprinting. 80% of iPPSD3 patients have methylation defect at GNAS A/B:TSS-DMR and at least one other GNAS DMR (most commonly including GNAS-AS1:TSS-DMR). They are known as sporadic cases. GNAS-miRNAs are located on the 3'UTR of the antisense transcript GNAS-AS1. We hypothesize that abnormal expression of the GNAS-miRNAs may contribute to the phenotype of iPPSD3 patients with methylation defect at GNAS-AS1:TSS-DMR. We used fibroblasts obtained from a patient with uniparental maternal disomy of chr. 20 (matUPD20) and leukocytes from a patient with paternal UPD20 (patUPD20) and from controls to measure expression of GNAS-miRNAs. In silico targets of GNAS-miRNAs have been characterized using miRbase. Finally, HEK cells were transfected with GNAS-miRNAs in order to identify targets. We found that GNAS-miRNAs are subjected to parental imprinting. In fact, hsa-miR-296-5p and hsa-miR-296-3p are under-expressed by 400-fold (p=0.0015) and 3.5-fold (p=0.0289) in the fibroblasts of the matUPD20 patient, respectively. We identified in silico, three groups of GNAS-miRNAs targets classified into cAMP signaling, calcium signaling and growth. Overexpression of hsa-miR-296-3p in HEK cells significantly represses PRKAG1 and GNB2 transcripts expression, p

The paradox of human rights is that they are usually only evidenced by their violation. The problem can generally be said to be ‘political,’ as it is hard to establish anything as ‘universal’ in a world divided into independent nation states. Of course, as long as law remains anchored in the state, it is also a ‘legal’ problem too. But systems theorists have pointed to an alternative in the ‘social positivisation’ of human rights, a way in which rights find positivisation through their actualisation in the decision-making and self-governance of the many special areas of society that now exist at the global level beyond the state. This paper examines the theory proposed in this respect by Gunter Teubner. It considers what is involved in Teubner’s concept of the social positivsation of human rights, before exploring the problems it faces in relation to the role of the state and the difficulties of positivising such highly generalised norms through such special social systems. The purpose, though, is not simply to rehash existing critiques of the concept, but to point out how the issues faced by this attempt to properly address the problem of human rights are a reflection of a general methodological development of systems theoretical approaches to law in recent years, whereby the focus has shifted excessively to ‘coding’ as the self-referential communication of social systems. It is argued that, whatever meaning they have in transnational regimes, human rights are important to meaning and communication at a most general social level and that this is missed by the excessive focus on specialised reflexivity and coding. The chapter will conclude by arguing that further insights into the problem of universal human rights, and the solutions that have emerged in response to it, are better gained by adopting a more functional perspective that focuses on human rights in world society as the general social system.

This Consensus Statement covers recommendations for the diagnosis and management of patients with pseudohypoparathyroidism (PHP) and related disorders, which comprise metabolic disorders characterized by physical findings that variably include short bones, short stature, a stocky build, early-onset obesity and ectopic ossifications, as well as endocrine defects that often include resistance to parathyroid hormone (PTH) and TSH. The presentation and severity of PHP and its related disorders vary between affected individuals with considerable clinical and molecular overlap between the different types. A specific diagnosis is often delayed owing to lack of recognition of the syndrome and associated features. The participants in this Consensus Statement agreed that the diagnosis of PHP should be based on major criteria, including resistance to PTH, ectopic ossifications, brachydactyly and early-onset obesity. The clinical and laboratory diagnosis should be confirmed by a molecular genetic analysis. Patients should be screened at diagnosis and during follow-up for specific features, such as PTH resistance, TSH resistance, growth hormone deficiency, hypogonadism, skeletal deformities, oral health, weight gain, glucose intolerance or type 2 diabetes mellitus, and hypertension, as well as subcutaneous and/or deeper ectopic ossifications and neurocognitive impairment. Overall, a coordinated and multidisciplinary approach from infancy through adulthood, including a transition programme, should help us to improve the care of patients affected by these disorders, This Consensus Statement and the series of consensus meetings were supported by funds from the European Cooperation in Science and Technology (COST) action BM1208 on imprinting disorders (www.imprinting- disorders.eu), the European Society for Paediatric Endocrinology (ESPE) and the European Society for Endocrinology (ESE). Travel costs and housing of the representatives of the Asian Pacific Paediatric Endocrine Society (APPES) and of the Pediatric Endocrine Society (PES) were supported by their societies. The authors received no funding from pharmaceutical companies

International audience; GIP-dependent Cushing's syndrome is caused by ectopic expression of glucose-dependent insulinotropic polypeptide receptor (GIPR) in cortisol-producing adrenal adenomas or in bilateral macronodular adrenal hyperplasias. Molecular mechanisms leading to ectopic GIPR expression in adrenal tissue are not known. Here we performed molecular analyses on adrenocortical adenomas and bilateral macronodular adrenal hyperplasias obtained from 14 patients with GIP-dependent adrenal Cushing's syndrome and one patient with GIP-dependent aldosteronism. GIPR expression in all adenoma and hyperplasia samples occurred through transcriptional activation of a single allele of the GIPR gene. While no abnormality was detected in proximal GIPR promoter methylation, we identified somatic duplications in chromosome region 19q13.32 containing the GIPR locus in the adrenocortical lesions derived from 3 patients. In 2 adenoma samples, the duplicated 19q13.32 region was rearranged with other chromosome regions, whereas a single tissue sample with hyperplasia had a 19q duplication only. We demonstrated that juxtaposition with cis-acting regulatory sequences such as glucocorticoid response elements in the newly identified genomic environment drives abnormal expression of the translocated GIPR allele in adenoma cells. Altogether, our results provide insight into the molecular pathogenesis of GIP-dependent Cushing's syndrome, occurring through monoallelic transcriptional activation of GIPR driven in some adrenal lesions by structural variations.

ObjectiveDisorders caused by impairments in the parathyroid hormone (PTH) signalling pathway are historically classified under the term pseudohypoparathyroidism (PHP), which encompasses rare, related and highly heterogeneous diseases with demonstrated (epi)genetic causes. The actual classification is based on the presence or absence of specific clinical and biochemical signs together with anin vivoresponse to exogenous PTH and the results of anin vitroassay to measure Gsa protein activity. However, this classification disregards other related diseases such as acrodysostosis (ACRDYS) or progressive osseous heteroplasia (POH), as well as recent findings of clinical and genetic/epigenetic background of the different subtypes. Therefore, the EuroPHP network decided to develop a new classification that encompasses all disorders with impairments in PTH and/or PTHrP cAMP-mediated pathway.Design and methodsExtensive review of the literature was performed. Several meetings were organised to discuss about a new, more effective and accurate way to describe disorders caused by abnormalities of the PTH/PTHrP signalling pathway.Results and conclusionsAfter determining the major and minor criteria to be considered for the diagnosis of these disorders, we proposed to group them under the term ‘inactivating PTH/PTHrP signalling disorder’ (iPPSD). This terminology: (i) defines the common mechanism responsible for all diseases; (ii) does not require a confirmed genetic defect; (iii) avoids ambiguous terms like ‘pseudo’ and (iv) eliminates the clinical or molecular overlap between diseases. We believe that the use of this nomenclature and classification will facilitate the development of rationale and comprehensive international guidelines for the diagnosis and treatment of iPPSDs.

Context: The term pseudohypoparathyroidism (PHP) was coined to describe the clinical condition resulting from end-organ resistance to parathormone (rPTH), caused by genetic and/or epigenetic alterations within or upstream of GNAS. Although knowledge about PHP is growing, there are few data on the prevalence of underlying molecular defects. Objective: The purpose of our study was to ascertain the relative prevalence of PHP-associated molecular defects. Design: With a specially designed questionnaire, we collected data from all patients (n = 407) clinically and molecularly characterized to date by expert referral centers in France, Italy, and Spain. Results: Isolated rPTH (126/407, 31%) was caused only by epigenetic defects, 70% of patients showing loss of imprinting affecting all four GNAS differentially methylated regions and 30% loss of methylation restricted to the GNAS A/B:TSS-DMR. Multihormone resistance with no Albright’s hereditary osteodystrophy (AHO) signs (61/407, 15%) was essentially due to epigenetic defects, although 10% of patients had point mutations. In patients with rPTH and AHO (40/407, 10%), the rate of point mutations was higher (28%) and methylation defects lower (about 70%). In patients with multihormone resistance and AHO (155/407, 38%), all types of molecular defects appeared with different frequencies. Finally, isolated AHO (18/407, 4%) and progressive osseous heteroplasia (7/407, 2%) were exclusively caused by point mutations. Conclusion: With European data, we have established the prevalence of various genetic and epigenetic lesions in PHP-affected patients. Using these findings, we will develop objective criteria to guide cost-effective strategies for genetic testing and explore the implications for management and prognosis.

Le syndrome de Cushing dependant de l’alimentation est du a l’expression illegitime du glucose-dependent insulinotropic peptide receptor (GIPR) dans les adenomes ou dans les hyperplasies surrenaliennes. Les mecanismes moleculaires conduisant a l’expression ectopique du GIPR dans les lesions surrenaliennes, qui conferent une sensibilite anormale au GIP, ne sont pas connus. Nous avons analyse 16 echantillons surrenaliens obtenus a partir de 14 patients presentant un syndrome de Cushing GIP-dependant et d’un patient avec un hyperaldosteronisme GIP-dependant. Le RNA FISH, permettant de visualiser l’ARN naissant dans les noyaux interphasiques, a mis en evidence une expression monoallelique du GIPR dans l’ensemble des echantillons. Le pyrosequencage n’a pas identifie d’anomalies de methylation du promoteur de GIPR. La CGH-array a identifie des duplications de la region 19q13.32 contenant le gene GIPR dans 3 echantillons. Le sequencage de nouvelle generation Mate-Pair et le ADN FISH ont montre dans 2 adenomes que les regions dupliquees etaient rearrangees avec d’autres regions chromosomiques. La juxtaposition avec des sequences regulatrices en cis, comme des elements de reponse aux glucocorticoides, dans ce nouvel environnement genomique conduit a l’expression anormale de l’allele transloque du GIPR dans les cellules adenomateuses et in vitro dans les cellules H295R. En conclusion, nous demontrons pour la premiere fois, que l’expression illegitime du GIPR dans les lesions surrenaliennes des patients avec syndrome de Cushing dependant de l’alimentation provient systematiquement d’un seul allele du gene. Cette expression monoallelique du GIPR est due dans certaines lesions a des rearrangements chromosomiques creant un nouvel environnement genomique favorable a son expression.

La PHP1B est caracterisee par une resistance a la PTH. La PHP1B est causee par des modifications epigenetiques dans une ou plusieurs regions differentiellement methylees (DMR) dans le locus GNAS. Dans la forme autosomique dominante (AD-PHP1B), les patients ont une perte d’empreinte (LOI) restreinte a la DMR GNAS-A/B et la plupart d’entre eux ont une deletion maternelle recurrente du gene STX16. Les patients sporadiques (sporPHP1B) ont une perte d’empreinte touchant GNAS. Recemment, Rochtus et al. ont identifie une nouvelle DMR dans GNAS (GNAS-AS2). Objectifs Identifier le profil de methylation du DMR GNAS-AS2 chez les patients atteints de PHP1B et caracteriser cette DMR. Patients AD-PHP1B, deletion STX16 (AD+ : n = 10) ; AD-PHP1B, pas de deletion STX16 (AD- : n = 4) ; sporPHP1B (n = 10) ; temoins (n = 10). Les deletions de STX16 et de GNAS ont ete exclues par MLPA, multiplex genomique et PCR quantitative. Resultats (1) Les AD− ont un index de methylation de la DMR AS2 significativement superieur, (38 %), a celui des temoins (22,7 %), des AD + (4 %) et des sporPHP1B (2,9 %) (p Conclusion Nous avons mieux caracterise la DMR GNAS-AS2 et identifie un sous-groupe de patients PHP1B (AD−) qui a un profil specifique de methylation de la DMR GNAS-AS2. Les AD− ont une LOI restreinte aux DMRs GNAS-A/B et GNAS-AS2. L’identification d’un profil specifique de methylation sur AS2 est en faveur d’une origine moleculaire commune pour ces patients.

Background GIP-dependent Cushing's syndrome is caused by ectopic expression of glucose-dependent insulinotropic polypeptide receptor (GIPR) in cortisol-producing adrenal adenomas or in bilateral macronodular adrenal hyperplasias. Molecular mechanisms leading to ectopic GIPR expression in adrenal tissue are not known. Methods We performed molecular analyses on 14 adrenal samples obtained from 12 patients with overt GIP-dependent Cushing's syndrome and from one patient with GIP-dependent aldosteronism. Results Nascent RNA FISH showed that GIPR expression in all adenoma and hyperplasia samples occurred through transcriptional activation of a single allele of the GIPR gene. While no abnormality was detected in proximal GIPR promoter methylation, we identified by CGH-array somatic duplications in chromosome region 19q13.32 containing the GIPR locus in the adrenocortical lesions derived from 3 patients. In 2 adenoma samples, the duplicated 19q13.32 region was rearranged with other chromosome regions, whereas a single tissue sample with hyperplasia had 19q duplication only. These duplications were further confirmed by DNA FISH. Among the 5 genes encompassed in the smallest region of overlap, only GIPR was over expressed. We demonstrated in vitro that juxtaposition with cis-acting regulatory sequences such as glucocorticoid response elements in the newly identified genomic environment drives abnormal expression of the translocated GIPR allele in HEK293 T and H295R cells. Conclusion Altogether, our results provide insight into the molecular pathogenesis of GIP-dependent Cushing's syndrome, occurring through monoallelic transcriptional activation of GIPR driven in some adrenal lesions by structural variations.

Les differents defauts moleculaires de GNAS causent les pseudohypoparathyroidies (PHP). La PHP1A et la PseudoPHP sont dues a des mutations maternelles et paternelles de la sequence codante. La PHP1B est due a une perte de methylation (PdM) du promoteur A/B de GNAS ; dans la forme dominante (AD-PHP1B), il s’y associe des deletions genomiques a proximite ou dans le locus GNAS ; dans la forme sporadique (sporPHP1B), la cause est inconnue. Objectifs Alors que la croissance fœtale est restreinte dans la PHP1A et la PseudoPHP, et excessive dans la PHP1B, nous proposons d’etudier la taille finale des differentes cohortes de PHP. Methodes Mediane (DS) des tailles finales (>18 ans) et IMC de 121 femmes (F) et 81 hommes (M) ; PHP1A (n = 72), PseudoPHP (n = 26), AD-PHP1B (n = 33) ou sporPHP1B (n = 71). Resultats Les PHP1A et PseudoPHP ont une petite taille finale [−2,8 (1,2) et −2,9 (1,5)], alors que les PHP1B ont une taille finale normale [sporPHP1B : −0,3 (1,1) et AD-PHP1B : −0,3 (1,4)]. Les PHP1A, mais pas les PseudoPHP, ont une obesite marquee a l’âge adulte 1,3 (1,4) vs 0,6 (1,7). Enfin, les femmes sporPHP1B sont significativement plus obeses que les hommes [1,2 vs 0,3 ; p = 0,0001]. Conclusion Nous decrivons pour la 1re fois : 1) que les PseudoPHP sont petits ; 2) que les PHP1B ont une taille normale malgre leur macrosomie a la naissance ; 3) que les femmes PHP1B ont une tendance a l’obesite, ce qui doit impliquer une surveillance metabolique.

La PHP1B est caracterisee par une perte d’empreinte du locus GNAS avec diminution de l’expression de Gsa, proteine cle de la signalisation AMPc. Les symptomes de la PHP1B ne sont pas expliques par le deficit de Gsa. Le transcrit GNAS-AS1 produit 3 miRNAs sur son extremite 3′UTR (hsa-miR-296-5p, hsa-miR-296-3p et hsa-mir-298-5p). Objectifs Demontrer que les GNAS-miRNAs sont soumis a empreinte, caracteriser leur expression et identifier leurs cibles. Methodes Pyrosequencage – qPCR – caracterisation in silico des cibles putatives des miRNAs – surexpression du hsa-miR-296-3p dans des cellules HEK293. Resultats Premierement, les GNAS-miRNAs sont soumis a empreinte (dans des fibroblastes de patient matUPD20, sous-expression du hsa-miR-296-5p et du hsa-miR-296-3p de 400 [ p = 0,0015] et de 3,5 [ p = 0,0289] fois) ; deuxiement, les cibles des miRNAs ont ete caracterisees in silico en utilisant miRBase et classes en trois groupes : signalisation de l’AMPc, signalisation du calcium et croissance ; troisiement, dans les cellules HEK, la surexpression du hsa-miR-296-3p reprime significativement l’expression d’AKAP6 et de PRKAG1 de 35 % ( p = 0,000085) et 44 % ( p = 0,000023). Dans les leucocytes d’un patient patUPD20, nous avons constate une diminution significative d’AKAP6 ( p = 0,0015) et une baisse non significative de l’expression de PRKAG1. Ces deux facteurs sont impliques dans la signalisation de l’AMPc ; quatriement, dans les HEK, la surexpression du hsa-miR-296-3p reprime significativement l’expression des 3 transcrits paternels de GNAS (GNAS-A/B, p = 0,00003 GNAS-AS1, p = 0,0007 et GNAS-Gsa, p = 0,0006). Discussion Les miRNAs de GNAS semblent soumis a l’empreinte parentale ; ils pourraient etre impliques dans la physiopathologie de la maladie.