Albright’s hereditary osteodystrophy (AHO) is a rare inherited disease characterized by skeletal abnormalities, short stature, and, in some cases, resistance to parathyroid hormone, resulting in pseudohypoparathyroidism (PHP). Heterozygous inactivating mutations of the GNAS1 gene are responsible for reduced activity of the alpha subunit of the Gs protein (GSα), a protein that mediates hormone signal transduction across cell membranes. Gsα is also known to have oncogenic potentials, leading to the development of human pituitary tumors and Leydig cell tumors. Here, we report the 1st case, a 3.5-year-old girl, with classic AHO phenotype and PHP type 1A associated with a cerebellar pilocytic astrocytoma. Coincidence or genetic relationships of both diseases are discussed according to molecular findings and current literature. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

Albright’s hereditary osteodystrophy (AHO) is a rare inherited disease characterized by skeletal abnormalities, short stature, and, in some cases, resistance to parathyroid hormone, resulting in pseudohypoparathyroidism (PHP). Heterozygous inactivating mutations of the GNAS1 gene are responsible for reduced activity of the alpha subunit of the Gs protein (GSα), a protein that mediates hormone signal transduction across cell membranes. Gsα is also known to have oncogenic potentials, leading to the development of human pituitary tumors and Leydig cell tumors. Here, we report the 1st case, a 3.5-year-old girl, with classic AHO phenotype and PHP type 1A associated with a cerebellar pilocytic astrocytoma. Coincidence or genetic relationships of both diseases are discussed according to molecular findings and current literature. / Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich.

INTRODUÇÃO: A primeira doença humana atribuída à resistência hormonal foi o pseudo-hipoparatireoidismo (PHP), uma doença rara caracterizada por hipocalcemia, hiperfosfatemia e níveis elevados de hormônio paratireoidiano (PTH) na presença de função renal normal, quadro condizente com resistência ao PTH. A classificação original do PHP leva em consideração a osteodistrofia hereditária de Albright (AHO): presente no PHP1a e ausente no PHP1b. Na medida em que as bases moleculares do PHP têm sido compreendidas, uma classificação baseada no genótipo tem surgido. Segundo ela, pacientes com PHP1a apresentam mutações na região codificadora da Gsalfa do GNAS e o padrão de herança é autossômico dominante relacionado à transmissão materna. Por outro lado, o PHP1b é caracterizado por alterações nas regiões diferencialmente metiladas (DMRs) do GNAS por mecanismos não completamente esclarecidos, limitando a previsão do seu padrão de herança. Pacientes que apresentam a AHO na ausência de resistência hormonal têm o diagnóstico de pseudopseudo-hipoparatireoidismo (PPHP) e seu padrão de herança é autossômico dominante relacionado à transmissão paterna de mutações na região codificadora da Gsalfa do GNAS. OBJETIVOS: Classificar 25 pacientes com PHP com base em defeitos no GNAS e caracterizar seu fenótipo. Pesquisar mutações no GNAS nos quatro pacientes com PPHP e também caracterizar seu fenótipo. MÉTODOS: A avaliação fenotípica incluiu análise das resistências hormonais, pesquisa de repercussões crônicas da hipocalcemia/hiperfosfatemia (calcificações em sistema nervoso central: SNC e catarata) e identificação da AHO. A análise do GNAS foi feita por sequenciamento automático e MLPA (região codificadora da Gsalfa) e por MS-MLPA (região regulatória: DMRs). RESULTADOS: Resistência ao PTH foi identificada nos 25 pacientes com PHP e resistência ao TSH em 17/25. Calcificações em SNC e catarata estiveram presentes em 18 e 10 pacientes com PHP, respectivamente. A AHO foi caracterizada por: face arredondada (n=18), braquidactilia (n=11), baixa estatura (n=8), ossificações subcutâneas (n=6), obesidade (n=9) e retardo mental (n=3). Identificamos oito mutações (cinco novas) na região codificadora da Gsalfa em 10 pacientes com PHP1a e quatro pacientes com PPHP. Quinze pacientes apresentaram alteração no padrão de metilação das DMRs (genótipo: PHP1b). O fenótipo dos pacientes foi semelhante nos dois grupos. DISCUSSÃO E CONCLUSÃO: Nenhuma das classificações do PHP foi capaz de predizer gravidade ou o curso clínico da doença. Porém, o diagnóstico do PHP1a baseado no genótipo possibilitou a identificação precoce de uma paciente, a exclusão de PHP1a na filha de outra paciente e o aconselhamento genético em duas famílias. O diagnóstico de PHP1b em uma paciente só foi possível graças ao genótipo, visto que seu perfil laboratorial osteometabólico era inconclusivo. Com base no fenótipo, 8/15 pacientes com PHP1b seriam classificados como PHP1a considerando a presença de dois ou mais estigmas da AHO, podendo levar a falhas no aconselhamento genético. Portanto, concluímos que a classificação do PHP baseada na análise do GNAS é mais informativa do que a baseada no fenótipo, permitindo o diagnóstico precoce e o aconselhamento genético de casos familiais de PHP1a. A identificação do PHP1b deve ser promissora na medida em que seus mecanismos de transmissão forem mais bem entendidos / BACKGROUND: The first human disease attributed to hormone resistance was pseudohypoparathyroidism (PHP), a rare disease characterized by hypocalcemia, hyperphosphatemia and elevated parathyroid hormone (PTH) levels in the presence of normal renal function, consistent picture of PTH resistance. The original classification of PHP takes into account the Albright hereditary osteodystrophy (AHO): present in PHP1a and absent in PHP1b. As the molecular bases of PHP have been understood, a classification based on genotype has emerged. According to it, PHP1a patients present mutations in the Gsalpha coding region of the GNAS and the pattern of inheritance is autosomal dominant related to maternal transmission. On the other hand, PHP1b is characterized by alterations in differentially methylated regions (DMRs) of the GNAS by mechanisms not completely clear, limiting the prediction of the pattern of inheritance. Patients who present AHO in the absence of hormone resistance have the diagnosis of pseudopseudohypoparathyroidism (PPHP) and their pattern of inheritance is autosomal dominant related to paternal transmission of mutations in the Gsalfa coding region of the GNAS. OBJECTIVE: To classify 25 patients with PHP based on GNAS molecular defects and to characterize their phenotype. To search for GNAS mutations in four patients with PPHP and also to characterize their phenotype. METHODS: The phenotypic evaluation included analysis of hormone resistances, research of chronic repercussions of hypocalcemia/hyperphosphatemia (calcifications in central nervous system: CNS and cataract) and identification of AHO. The analysis of the GNAS was done by automated sequencing and MLPA (Gsalphaa coding region) and by MS-MLPA (regulatory region: DMRs). RESULTS: PTH resistance was identified in 25 patients with PHP and TSH resistance in 17/25. Calcifications in CNS and cataract were present in 18 and 10 patients with PHP, respectively. AHO was characterized by: rounded face (n=18), brachydactyly (n=11), short stature (n=8), subcutaneous ossifications (n=6), obesity (n=9) and mental retardation (n=3). We identified eight mutations (five novels) in the Gsalpha coding region in 10 patients with PHP1a. Fifteen patients presented alterations in the methylation pattern of DMRs (genotype: PHP1b). The phenotype of patients was similar in both groups. DISCUSSION AND CONCLUSION: None of the PHP classifications was able to predict the severity or clinical course of the disease. However, the diagnosis of PHP1a based on genotype allowed the early identification of one patient, the exclusion of PHP1a in the daughter of another patient and genetic counseling in two families. The PHP1b diagnosis in one patient was only possible due to the genotype, as her bone metabolism profile was inconclusive. Based on phenotype, 8/15 PHP1b patients would have been classified as PHP1a considering the presence of two or more AHO stigmas, being able to lead to failures in genetic counseling. Therefore, we conclude that the PHP classification based on GNAS analysis is more informative than that based on phenotype, allowing the early diagnosis and the genetic counseling for familial cases of PHP1a. The identification of PHP1b may be promising as its transmission mechanisms are better clarified

GNAS est un locus complexe soumis à l'empreinte parentale. Il code pour cinq transcrits alternatifs dont l’expression est régulée de manière parentale, tissulaire et développementale : la sous-Unité alpha stimulatrice de la protéine G hétérotrimérique (Gαs), XLαs, NESP55, et deux ARNnc, A/B et GNAS-AS1. Gαs est une protéine clé dans la transduction hormonale partageant avec XLαs la capacité de produire l'AMPc intracellulaire après stimulation des récepteurs couplés à Gαs.Dans la première partie de ma thèse, je me suis concentrée sur l'étude du rôle des transcrits de GNAS, en particulier XLαs, dans la croissance fœtale et post-Natale. J’ai profité du modèle unique des pseudohypoparathyroïdies (PHPs), pathologies humaines rare de l’empreinte, causées par des anomalies génétiques ou épigénétiques du locus GNAS altérant le dosage génique des transcrits de GNAS. La croissance anormale est une caractéristique majeure des PHPs.Dans la deuxième partie de ma thèse, j’ai étudié le profil épigénétique du locus GNAS (méthylation de l'ADN et expression des transcrits) dans les cellules souches humaines embryonnaires -HESCs-, dans les cellules pluripotentes induites dérivées à partir de fibroblastes de sujets sains -IPSCs- et dans les cellules redifférenciées en cellules souches neurales et mésenchymateuses. La caractérisation précise du locus humain GNAS en physiologie (cellules souches) et pathologie (PHP) est essentielle pour une meilleure compréhension des processus développementaux importants comme la croissance. L'exploration du phénotype "croissance" de différents types de PHPs a permis de mieux comprendre le rôle des transcrits du locus GNAS dans la physiologie et la physiopathologie. L'analyse de cellules des PHPs a permis de mieux caractériser l’impact des anomalies moléculaires du locus GNAS en pathologie humaine. Les hiPSCs peuvent être un outil utile pour étudier les modifications épigénétiques au niveau du locus GNAS. / GNAS is a complex locus subjected to parental imprinting encoding five parental-, tissue- and developmental-Manner regulated transcripts : the alpha stimulatory subunit of the G protein (Gαs), XLαs, NESP55, and two ncRNAs, A/B and the antisens GNAS-AS1. Gαs is a key protein in hormonal signaling sharing with XLαs the ability to produce intracellular cAMP upon stimulation of Gαs-Coupled receptors. In the first part of my thesis, I focused on studying the role of the GNAS transcripts, particularly XLαs, in fetal and postnatal growth. I took advantage of the unique model of pseudohypoparathyroidism (PHP), a rare human disease, caused by genetic or epigenetic abnormalities at the GNAS locus leading to various combinations of GNAS transcripts alterations. Abnormal growth appears to be a major feature of PHP. In the second part of my thesis, I studied the epigenetic pattern of GNAS (DNA methylation and transcripts expression) in human embryonic stem cells -HESCs-, in induced pluripotent stem cells -IPSCs- derived from fibroblasts from healthy individuals, and in cells re-Differentiated from these stem cells in neuronal and mesenchymal cells. The precise characterization of the human GNAS locus in physiology (stem cells) and pathology (PHP) is critical for a better understanding of major processes like growth. Through exploration of the "growth" phenotype of different groups of PHPs we have participated to the better understanding of the role of the GNAS transcripts in the physiology and pathophysiology. Human iPSCs may be an useful tool to study epigenetic modifications at the GNAS locus.

INTRODUÇÃO: A primeira doença humana atribuída à resistência hormonal foi o pseudo-hipoparatireoidismo (PHP), uma doença rara caracterizada por hipocalcemia, hiperfosfatemia e níveis elevados de hormônio paratireoidiano (PTH) na presença de função renal normal, quadro condizente com resistência ao PTH. A classificação original do PHP leva em consideração a osteodistrofia hereditária de Albright (AHO): presente no PHP1a e ausente no PHP1b. Na medida em que as bases moleculares do PHP têm sido compreendidas, uma classificação baseada no genótipo tem surgido. Segundo ela, pacientes com PHP1a apresentam mutações na região codificadora da Gsalfa do GNAS e o padrão de herança é autossômico dominante relacionado à transmissão materna. Por outro lado, o PHP1b é caracterizado por alterações nas regiões diferencialmente metiladas (DMRs) do GNAS por mecanismos não completamente esclarecidos, limitando a previsão do seu padrão de herança. Pacientes que apresentam a AHO na ausência de resistência hormonal têm o diagnóstico de pseudopseudo-hipoparatireoidismo (PPHP) e seu padrão de herança é autossômico dominante relacionado à transmissão paterna de mutações na região codificadora da Gsalfa do GNAS. OBJETIVOS: Classificar 25 pacientes com PHP com base em defeitos no GNAS e caracterizar seu fenótipo. Pesquisar mutações no GNAS nos quatro pacientes com PPHP e também caracterizar seu fenótipo. MÉTODOS: A avaliação fenotípica incluiu análise das resistências hormonais, pesquisa de repercussões crônicas da hipocalcemia/hiperfosfatemia (calcificações em sistema nervoso central: SNC e catarata) e identificação da AHO. A análise do GNAS foi feita por sequenciamento automático e MLPA (região codificadora da Gsalfa) e por MS-MLPA (região regulatória: DMRs). RESULTADOS: Resistência ao PTH foi identificada nos 25 pacientes com PHP e resistência ao TSH em 17/25. Calcificações em SNC e catarata estiveram presentes em 18 e 10 pacientes com PHP, respectivamente. A AHO foi caracterizada por: face arredondada (n=18), braquidactilia (n=11), baixa estatura (n=8), ossificações subcutâneas (n=6), obesidade (n=9) e retardo mental (n=3). Identificamos oito mutações (cinco novas) na região codificadora da Gsalfa em 10 pacientes com PHP1a e quatro pacientes com PPHP. Quinze pacientes apresentaram alteração no padrão de metilação das DMRs (genótipo: PHP1b). O fenótipo dos pacientes foi semelhante nos dois grupos. DISCUSSÃO E CONCLUSÃO: Nenhuma das classificações do PHP foi capaz de predizer gravidade ou o curso clínico da doença. Porém, o diagnóstico do PHP1a baseado no genótipo possibilitou a identificação precoce de uma paciente, a exclusão de PHP1a na filha de outra paciente e o aconselhamento genético em duas famílias. O diagnóstico de PHP1b em uma paciente só foi possível graças ao genótipo, visto que seu perfil laboratorial osteometabólico era inconclusivo. Com base no fenótipo, 8/15 pacientes com PHP1b seriam classificados como PHP1a considerando a presença de dois ou mais estigmas da AHO, podendo levar a falhas no aconselhamento genético. Portanto, concluímos que a classificação do PHP baseada na análise do GNAS é mais informativa do que a baseada no fenótipo, permitindo o diagnóstico precoce e o aconselhamento genético de casos familiais de PHP1a. A identificação do PHP1b deve ser promissora na medida em que seus mecanismos de transmissão forem mais bem entendidos / BACKGROUND: The first human disease attributed to hormone resistance was pseudohypoparathyroidism (PHP), a rare disease characterized by hypocalcemia, hyperphosphatemia and elevated parathyroid hormone (PTH) levels in the presence of normal renal function, consistent picture of PTH resistance. The original classification of PHP takes into account the Albright hereditary osteodystrophy (AHO): present in PHP1a and absent in PHP1b. As the molecular bases of PHP have been understood, a classification based on genotype has emerged. According to it, PHP1a patients present mutations in the Gsalpha coding region of the GNAS and the pattern of inheritance is autosomal dominant related to maternal transmission. On the other hand, PHP1b is characterized by alterations in differentially methylated regions (DMRs) of the GNAS by mechanisms not completely clear, limiting the prediction of the pattern of inheritance. Patients who present AHO in the absence of hormone resistance have the diagnosis of pseudopseudohypoparathyroidism (PPHP) and their pattern of inheritance is autosomal dominant related to paternal transmission of mutations in the Gsalfa coding region of the GNAS. OBJECTIVE: To classify 25 patients with PHP based on GNAS molecular defects and to characterize their phenotype. To search for GNAS mutations in four patients with PPHP and also to characterize their phenotype. METHODS: The phenotypic evaluation included analysis of hormone resistances, research of chronic repercussions of hypocalcemia/hyperphosphatemia (calcifications in central nervous system: CNS and cataract) and identification of AHO. The analysis of the GNAS was done by automated sequencing and MLPA (Gsalphaa coding region) and by MS-MLPA (regulatory region: DMRs). RESULTS: PTH resistance was identified in 25 patients with PHP and TSH resistance in 17/25. Calcifications in CNS and cataract were present in 18 and 10 patients with PHP, respectively. AHO was characterized by: rounded face (n=18), brachydactyly (n=11), short stature (n=8), subcutaneous ossifications (n=6), obesity (n=9) and mental retardation (n=3). We identified eight mutations (five novels) in the Gsalpha coding region in 10 patients with PHP1a. Fifteen patients presented alterations in the methylation pattern of DMRs (genotype: PHP1b). The phenotype of patients was similar in both groups. DISCUSSION AND CONCLUSION: None of the PHP classifications was able to predict the severity or clinical course of the disease. However, the diagnosis of PHP1a based on genotype allowed the early identification of one patient, the exclusion of PHP1a in the daughter of another patient and genetic counseling in two families. The PHP1b diagnosis in one patient was only possible due to the genotype, as her bone metabolism profile was inconclusive. Based on phenotype, 8/15 PHP1b patients would have been classified as PHP1a considering the presence of two or more AHO stigmas, being able to lead to failures in genetic counseling. Therefore, we conclude that the PHP classification based on GNAS analysis is more informative than that based on phenotype, allowing the early diagnosis and the genetic counseling for familial cases of PHP1a. The identification of PHP1b may be promising as its transmission mechanisms are better clarified