O sistema da procineticina desempenha um papel importante na migração dos neurônios secretores de GnRH e na neurogênese do bulbo olfatório. Camundongos com ablação dos genes que codificam a procineticina 2 (PROK2) e seu receptor (PROKR2) apresentaram fenótipos semelhantes ao da síndrome de Kallmann descrita em humanos. Mutações inativadoras nos genes PROK2 e PROKR2 foram identificadas em pacientes com hipogonadismo hipogonadotrófico isolado. Com base nestes achados, investigamos a presença de alterações estruturais nos genes PROK2 e PROKR2 em 107 pacientes brasileiros (63 com síndrome de Kallmann e 47 com hipogonadismo hipogonadotrófico isolado normósmico). Cem indivíduos brasileiros que relataram desenvolvimento puberal normal foram utilizados como grupo controle. As regiões codificadoras dos genes PROK2 e PROKR2 foram amplificadas utilizando-se oligonucleotídeos intrônicos específicos, seguida de purificação enzimática e sequenciamento automático. Duas mutações no gene PROK2 foram identificadas: a mutação p.G100fsX121 em homozigose presente em dois irmãos com síndrome de Kallmann; e a mutação p.I55fsX56 em heterozigose identiificada em um paciente com HHIn. Quatro mutações foram identificadas no gene PROKR2 (p.R80C, p.Y140X, p.L173R e p.R268C) em cinco pacientes com síndrome de Kallmann e um paciente com HHIn. Essas mutações não foram encontradas no grupo controle. As mutações do tipo missense, p.R80C, p.L173R e p.R268C foram identificadas em heterozigose. Mutações nos genes FGFR1, GnRHR, KiSS-1 e GPR54 foram excluídas nesses pacientes. O paciente portador da mutação p.R268C do PROKR2 apresentou deleção dos exons 1 e 2 do gene KAL1. Adicionalmente, as mutações p.R80C e p.R268C foram identificadas em heterozigose em parentes de primeiro grau assintomáticos dos casos índices. A nova mutação p.Y140X do PROKR2, única alteração em homozigose, foi identificada em um paciente com micropênis, criptorquidia bilateral, anosmia e palato ogival. Os pais deste paciente eram portadores da mutação p.Y140X em heterozigose e relataram desenvolvimento puberal normal e ausência de anormalidades olfatórias. Estudos in vitro da nova mutação p.R80C localizada na primeira alça intracelular demonstraram que o acúmulo de fofatidil-inositol (IP), assim como a ativação da via da MAPK foram significativamente afetadas em células transfectadas com o receptor mutado em relação ao receptor selvagem, indicando que a mutação p.R80C determina uma menor atividade do receptor. Avaliação da expressão por Western blot mostrou uma diminuição na expressão do receptor mutado R80C e uma maior expressão de receptores imaturos. Esses achados sugeriram o papel crítico da arginina localizada na posição 80 na atividade normal do receptor. Em conclusão, expandimos o repertório de mutações deletérias nos genes PROK2 e PROKR2 em pacientes com hipogonadismo hipogonadotrófico isolado. A haploinsuficiência do PROKR2 não foi suficiente para causar síndrome de Kallmann ou HHIn, entretanto mutações inativadoras em homozigose nos genes PROK2 e PROKR2 foram responsáveis pelo fenótipo reprodutivo e olfatório anormal, em concordância com os estudos prévios de ablação gênica em modelos animais. Arginina localizada na posição 80 do PROKR2 desempenha um papel crucial na adequada maturação do receptor / Physiological activation of the prokineticin pathway has a critical role in olfactory bulb morphogenesis and GnRH secretion. Knock-out mice for genes that encode prokineticin 2 (PROK2) and the prokineticin receptor 2 (PROKR2) exhibited a phenotype similar to the Kallmann syndrome (KS). Inactivating mutations in PROK2 and PROKR2 have been identified in patients with isolated hypogonadotropic hypogonadism. Based on these findings, we investigated the presence of inactivating mutations of the genes PROK2 and PROKR2 in Brazilian patients with isolated hypogonadotropic hypogonadism associated or not with olfactory abnormalities and performed in vitro studies of the new identified mutations. We studied 107 patients with HH (63 with Kallmann syndrome and 44 with normosmic HH) and 100 control individuals. The coding regions of PROK2 and PROKR2 were amplified by polymerase chain reaction followed by enzymatic purification and direct automatic sequencing. In PROK2, two known frameshift mutations were identified. Two brothers with Kallmann syndrome harbored the homozygous p.G100fsX121 mutation, whereas one male with normosmic HH harbored the heterozygous p.I55fsX56 mutation. In PROKR2, four distinct mutations (p.R80C, p.Y140X, p.L173R and p.R268C) were identified in five patients with Kallmann syndrome and in one patient with normosmic HH. These mutations were not found in the control group. The p.R80C and p.R268C missense mutations were identified in heterozygous state in the HH patients and in their asymptomatic first-degree relatives. The p.L173R was also identified in heterozygous state. In addition, no mutations of FGFR1, GnRHR, KiSS-1 or GPR54 were identified in these patients. The patient with the PROKR2 mutation p.R268C also has a deletion of the exon 1 and 2 in the gene KAL1. Notably, the new nonsense mutation (p.Y140X) was identified in homozygous state in an anosmic boy with micropenis, bilateral cryptorchidism and high-arched palate. His asymptomatic parents were heterozygous for this severe defect. In vitro studies of the new mutation, p.R80C, were performed in order to access the mechanism by which this mutation could affect the activity of the PROKR2. In vitro studies showed that the amount of fofatidil-inositol (PI) and the activation of MAPK were significantly lower in cells transfected with the R80C mutant receptor than in cells transfected with the wild receptor, indicating that this variant is a loss-of-function mutation. Binding studies and Western blot showed a reduction in the expression levels of the receptor in the plasma membrane and in whole cell, respectively. Additionally, Western blot analysis of R80C PROKR2 revealed an additional smaller molecular weight band that represents the presence of immature unglycosylated receptors. The arginine 80 in ICL1 is important for post-translational processing of PROKR2. In conclusion, we expanded the repertoire of PROK2 and PROKR2 mutations in patients with HH and showed that PROKR2 haploinsufficiency is not sufficient to cause Kallmann syndrome or normosmic HH, whereas homozygous loss-of-function mutations either in PROK2 or PROKR2 are sufficient to cause disease phenotype, in accordance with the Prokr2 and Prok2 knockout mouse models. In vitro studies suggested that the arginine located at position 80 of the receptor seems to play an important role in the receptor function

Le syndrome CHARGE est une association malformative rare due à une mutation du gène CHD7 dans 60 à 90% des cas. L'objectif de ce travail était d'en décrire les éléments cliniques et moléculaires afin d'optimiser la prise en charge de patients atteints d'un handicap multisensoriel lourd.Le diagnostic anténatal en est difficile et l'étude de 40 fœtus a permis d'affiner la description du phénotype, de décrire de nouveaux éléments cliniques et finalement de proposer un ajustement des critères diagnostiques chez le fœtus.L'étude endocrinienne de 42 patients confirme la présence d'un hypogonadisme hypogonadotrope dans 97% des cas. Souvent méconnu et non traité il peut avoir des conséquences délétères sur la qualité de vie. Nous proposons qu'il soit reconnu comme critère majeur du syndrome.L'étude clinique d'une cohorte française de 119 patients a montré que la surdité et l'atteinte des canaux semi circulaires sont les éléments les plus fréquents du syndrome (suivis de l'atteinte hypophysaire, de l'arhinencéphalie et des anomalies de l'oreille externe), et les seuls significativement associés à la présence d'une mutation dans CHD7. Une étude approfondie des données issues de cette étude est en cours.Sur le plan moléculaire, alors que la plupart des mutations identifiées sont des mutations tronquantes privées apparues de novo, nous avons identifié 4 variants au niveau de l'intron 25, récurrents pour certains, dont l'interprétation était délicate. Leur étude in silico puis par une technique de minigène a permis de mettre en évidence une configuration particulière des séquences impliquées dans l'épissage de l'exon 26 (point de branchement distant) et de démontrer leur pathogénicité. / CHARGE syndrome is a rare disorder of multiple congenital anomalies ascribed to a CHD7 gene mutation in 60% to 90% of cases. The aim of this study was to improve the knowledge regarding molecular and clinical aspects of the syndrome in order to optimize the management of these patients with severe disability. Antenatal diagnosis remains challenging in many instances and a detailed clinicopathological survey in a series of 40 fetuses allowed us to refine the clinical description of CHARGE syndrome in fetuses, describe some novel features and set up diagnostic criteria. An endocrinologic study of 42 patients showed a hypogonadotrophic hypogonadism in 97% of cases. For this reason, it should be considered as a major symptom of the syndrome. An early screening should lead to a hormonal replacement therapy which dramatically impacts the condition.A study of a French cohort of 119 patients found that deafness and semi-circular canals hypoplasia were the most frequent symptoms (followed by hypogonadotrophic hypogonadism, arhinencephaly and external ears anomalies) and the only features statistically associated with a mutation in the CHD7 gene. A detailed study of the data is still going on.The syndrome is mainly due to de novo and private truncating mutations of the CHD7 gene but we report an intriguing hot spot of intronic mutations located in IVS25. Combining computational in silico analysis and ex vivo minigene assays, we explained this mutation hot spot by a particular genomic context, including a distant branch point, and confirmed the pathogenicity of these mutations.

A resposta do LH e do FSH ao estímulo com GnRH, realizado em estádio pré-puberal em pacientes com hipopituitarismo acompanhados até a idade puberal, são úteis para predizer o diagnóstico da deficiência de gonadotrofinas, principalmente nas meninas. O estudo da região codificadora do gene LH em pacientes com hipogonadismo hipogonadotrófico e concentrações normais de LH revelou 5 variantes alélicas. A freqüência das variantes alélicas Arg8 e Thr15 foi similar entre hipogonádicos e adultos normais e a sua presença não interferiu nas concentrações séricas do LH. O estudo das isoformas do LH mostrou um predomínio das isoformas ácidas do LH em hipogonádicos e indivíduos normais, não permitindo atribuir à sua presença a baixa atividade biológica do LH imunorreativo encontrado em 13% dos hipogonádicos / LH and FSH responses to GnRH stimulation carried out in the pre-pubertal stage in patients with hypopituitarism followed until the pubertal stage are useful tools for predicting the gonadotropin deficiency diagnosis, especially in girls. The study of the codifying region of the LH gene in patients with hypogonadotropic hypogonadism and normal LH levels disclosed 5 allelic variants. The frequencies of the allelic variants Arg8 and Thr15 were similar between hypogonadic and normal adults, and their presence did not alter serum LH levels. The study of LH isoforms showed a predominance of acid LH isoforms in hypogonadic and normal subjects, which does not allow us to ascribe to their presence the low biological activity of the immunoreactive LH, found in 13% of the hypogonadic individuals

O mecanismo de controle da secreção de GnRH inclui diversas vias neuronais. Estudos em modelos animais identificaram genes que codificam fatores de transcrição, tais como TTF-1 (thyroid transcription factor 1) e EAP1 (enhanced at puberty), que atuam no controle transcricional de genes codificadores de fatores excitatórios (KiSS1 e GnRH) e inibitórios (preproencefalinas) regulando a secreção de GnRH. Em primatas, a expressão de EAP1 e TTF-1 aumenta, no início da puberdade, nas regiões hipotalâmicas envolvidas na secreção de GnRH. Nos modelos animais, a deleção pós-natal de TTF-1 e o silenciamento do EAP1 provocam atraso puberal e prejuízo na função reprodutiva. TTF-1 também está envolvido na morfogênese diencefálica, por meio da via de sinalização da família Sonic-Hedgehog. Anormalidades na secreção de GnRH resultam em distúrbios puberais, que variam de puberdade precoce central (PPC) a hipogonadismo hipogonadotrófico. Hipotetizamos que anormalidades genéticas no TTF-1 e EAP1 estejam envolvidas na patogênese dos distúrbios puberais centrais. A PPC pode ser idiopática ou devido a causas orgânicas, sendo o hamartoma hipotalâmico, uma malformação congênita não neoplásica, a mais conhecida. Os pacientes com PPC devido a hamartoma hipotalâmico podem cursar com alterações neurológicas e cognitivas. Nossos objetivos foram: estudar as regiões codificadora do TTF-1 e do EAP1 e a região promotora do TTF-1 em pacientes com distúrbios puberais centrais; estabelecer a prevalência, taxa de penetrância e modo de herança da forma familial de PPC e caracterizar as manifestações neurológicas e neurocognitivas de pacientes com PPC devido a hamartoma hipotalâmico. Foram selecionados 133 pacientes com distúrbios puberais centrais - PPC idiopática (n=71), PPC devido a hamartoma hipotalâmico (n=15) e hipogonadismo hipogonadotrópico isolado normósmico (HHIn) (n=47) - e controles (n=53). Os genes TTF-1 e EAP1 foram amplificados e submetidos a sequenciamento automático. Os tratos de poliglutamina e polialanina no EAP1 foram estudados por software de análise de tamanho de fragmento (GeneScan). A avaliação neurológica e neurocognitiva dos pacientes com PPC devido a hamartoma hipotalâmico consistiu de exame neurológico, eletroencefalograma, ressonância magnética de encéfalo e aplicação da escala de inteligência (WISC-III, WAIS-III, WPPSIR). Identificamos 25% de casos familiais de PPC, com modo de herança autossômica dominante e taxa de penetrância de 67,5%. Variantes alélicas no TTF-1 não foram identificadas nos pacientes estudados. No gene EAP1 foram identificadas quatro variantes alélicas sinônimas: p.E87E, p.A163A, p.Y415Y e uma nova variante alélica p.C758C, encontradas em pacientes com PPC e HHIn. A distribuição das frequências alélica e genotípica das variantes alélicas do EAP1 não diferiram entre pacientes com PPC, HHIn e controles (p >0,05). Nas regiões poliglutamina e polialanina 5 distal foi identificada variação similar no número de repetições glutamina e alanina em pacientes e controles. Não houve diferença significativa da frequência alélica em relação ao número de repetições glutamina e alanina entre os grupos PPC e HHIn (p >0,05). A avaliação neurológica dos pacientes com PPC devido a hamartoma hipotalâmico revelou epilepsia gelástica e crises focais com generalização em 3/15 (20%) pacientes. Não houve diferença significativa entre a mediana do maior diâmetro dos hamartomas dos pacientes com PPC com e sem epilepsia (13 e 10 mm, respectivamente). Quanto à forma, 10 hamartomas eram sésseis e 5 pedunculados, sendo que a forma pedunculada foi detectada exclusivamente em pacientes sem epilepsia. A avaliação neurocognitiva em 11 dos 15 pacientes com PPC devido a hamartoma hipotalâmico detectou 2 pacientes com epilepsia com QI significativamente menor que o grupo sem epilepsia (p <0,05). Em conclusão, (i) a considerável prevalência de casos familiais de PPC reforça a influência dos fatores genéticos na puberdade humana; (ii) mutações germinativas no TTF-1 e no EAP1 não estão envolvidas na patogênese dos distúrbios puberais centrais; (iii) a função neurocognitiva reduzida nos pacientes com hamartoma e epilepsia sugere um efeito deletério das crises convulsivas no sistema nervoso central / GnRH secretion control involves multiple neuronal pathways. Animal studies have identified genes which codifies transcription factors, such as TTF-1 (thyroid transcription factor 1) and EAP1 (enhanced at puberty), that act in the transcriptional control of genes that codifies excitatory (KiSS1 and GnRH) and inhibitory factors (preproenkephalines) regulating GnRH secretion. In nonhuman primates, expression of EAP1 and TTF-1 are increased at the hypothalamic regions involved in GnRH secretion, at the beginning of puberty. In animal models, post-natal TTF-1 deletion and silencing of EAP1 lead to pubertal delay and damage of reproductive function. TTF-1 is also involved in diencephalic morphogenesis, through signalization via Sonic-Hedgehog family. Abnormalities in GnRH secretion are responsible for pubertal disorders, varying from central precocious puberty (CPP) to hypogonadotropic hypogonadism. We hypothesized that genetic anomalies at TTF-1 and EAP1 are involved in the pathogenesis of central pubertal disorders. CPP may be idiopathic or due to organic alterations and hypothalamic hamartoma, a non-neoplasic congenital malformation, is the most frequent known organic cause. Patients with CPP due to hypothalamic hamartoma may have neurological and cognitive disfunctions. Our aims were: to evaluated the codifying region of TTF-1 and EAP1 and the promoter region of TTF-1 in patients with central pubertal disorders; to establish the prevalence, penetrance rate and inheritance mode of familial CPP and to characterize neurologic and neurocognitive aspects of patients with CPP due to hypothalamic hamartoma. We selected 133 patients with central pubertal disorders idiopathic CPP (n=71), CPP due to hypothalamic hamartoma (n=15) and normosmic isolated hypogonadropic hypogonadism (nIHH) (n=47) - and controls (n=53). TTF-1 and EAP1 genes were amplified and sequenced. Polyglutamine and polyalanine tracts of EAP1 were studied by a fragment size analyser software (GeneScan). Neurologic and neurocognitive evaluation of CPP patients due to hypothalamic hamartoma consisted of neurologic exam, electroencephalogram, brain magnetic resonance and application of intelligence scale (WISC-III, WAIS-III, WPPSI-R). We identified 25% of familial CPP cases with autosomal dominant mode of inheritance and penetrance rate of 67.5%. No TTF-1 allelic variants were identified in the patients analysed. At EAP1 gene, four synonimous allelic variants were identified: p.E87E, p.A163A, p.Y415Y and a new allelic variant p.C758C, found in CPP and nIHH patients. The allelic and genotypic distribution of theses variants of EAP1 did not differ among patients with CPP and nIHH, and controls (p >0.05). At polyglutamine and 5 distal polyalanine region, similar glutamine and alanine repeats variation was found. No significative difference of allelic frequency distribution regarding the number of glutamines and alanines repeats was found among the studied groups (p >0.05). Neurologic evaluation of CPP patients due to hypothalamic hamartoma revealed epilepsy and focal crisis with generalization in 3/15 (20%) of the patients. No significant difference between the median of the larger diameter of hypothalamic hamartoma of CPP patients with and without epilepsy was found (10 mm and 13 mm, respectively). Regarding the shape, 10 hamartomas were sessile and 5 pedunculated, and the pedunculated shape was found only in non epileptic patients. Neurocognitive evaluation performed in 11 of the 15 patients with CPP due to hypothalamic hamartoma detected 2 patients with epilepsy whose IQ were significantly lower than the IQ found in the group without epilepsy (p <0.05). In conclusion, (i) the considerable prevalence of familial CPP cases reinforce the influence of genetic factors in human puberty; (ii) germinative mutations in TTF-1 and EAP1 are not involved in the pathogenesis of central pubertal disorders; (iii) reduced neurocognitive function in patients with hypothalamic hamartoma and epilepsy suggests a deleterious effect of crisis at the central nervous system

Introdução. A insuficiência cardíaca (IC) é caracterizada por exacerbação da atividade nervosa simpática muscular (ANSM), baixa tolerância ao esforço e dispneia. Além disso, é característico nessa população o desequilíbrio entre o anabolismo e catabolismo, favorecendo dessa maneira uma acentuada perda da massa magra muscular, o que agrava ainda mais a qualidade de vida nos pacientes com IC. Dentre as alterações anabólicas observadas na IC avançada destaca-se a diminuição dos hormônios GH, IGF-1 e testosterona. A testosterona, um importante hormônio para as características masculinizantes e na manutenção da massa muscular, apresenta acentuada redução com o avançar da doença. Esta perda da massa magra, leva ao processo de caquexia muscular e consequente atrofia, com diminuição da força e da capacidade funcional do paciente com IC. A reposição de testosterona nesses pacientes tem sido estudada e se mostra uma importante terapêutica para melhorar a capacidade funcional e força muscular. Porém, não se conhece claramente o papel deste tratamento medicamentoso sobre o processo anabólico muscular, bem como na melhora da composição corporal. O exercício físico como tratamento não medicamentoso tem sido amplamente recomendado na IC por reduzir a ANSM, melhorar o fluxo sanguíneo periférico, aumentar a força muscular e melhorar a qualidade de vida. Entretanto, a combinação das estratégias do exercício físico associado à terapia de reposição de testosterona, não é conhecido em pacientes com IC. Métodos. 24 pacientes com IC foram randomizados em 3 grupos: Treinamento (TR, n=9), Testosterona (T, n=8) e Treino+Testosterona (TRT, n=7). A ANSM foi avaliada pela técnica de Microneurografia. O fluxo sanguíneo do antebraço foi avaliado pela pletismografia de oclusão venosa. A composição corporal foi avaliada pela densitometria (DEXA). A biópsia do músculo vasto-lateral foi feita para avaliarmos a área de secção transversa da fibra e a tipagem de fibras musculares. A qualidade de vida foi avaliada pelo questionário de Minnesota. O treinamento físico aeróbio em bicicleta foi realizado 3 vezes por semana, com 40 minutos de exercício por sessão, pelo período de 4 meses. A reposição de testosterona foi realizada pela administração intramuscular de undecilato de testosterona pelo período de 4 meses. Resultados. Após 4 meses de intervenção, observamos restauração dos níveis de testosterona em todos os grupos. A ANSM reduziu nos grupos TR e TRT. Não houve aumento do fluxo sanguíneo entre os grupos. O consumo de oxigênio aumentou em todos os grupos, porém apenas o grupo TRT aumentou a potência máxima ao exercício. A massa magra apresentou aumento significativo apenas no grupo TRT. Não observamos mudança no conteúdo mineral ósseo entre os grupos. Apenas o grupo TRT aumentou de maneira significativa a área de secção transversa das fibras tipo I (oxidativas). A qualidade de vida melhorou apenas nos grupos TR e TRT. Conclusões. O exercício físico associado à terapia de reposição de testosterona se mostrou mais eficaz em reduzir a ANSM, aumentar a capacidade funcional, a força muscular, a massa magra com um importante aumento das fibras do tipo I. Nossos resultados enfatizam a importância do exercício físico em pacientes com IC e traz uma nova perspectiva com a associação da testosterona para pacientes com hipogonadismo / Introduction. Heart failure (HF) is characterized by exacerbation of muscle sympathetic nerve activity (MSNA), exercise intolerance and dyspnea. Furthermore, is characteristic in this population the imbalance between anabolism and catabolism which lead to loss of skeletal muscle mass worsening quality of life in HF patients. Prior studies have demonstrated decrease in anabolic hormones such as GH, IGF-1 and testosterone. Testosterone, an important hormone for masculinization feature and maintenance of muscle mass, shows sharp decline in advanced HF. Loss muscle mass leads to cachexia and atrophy which decrease strength and functional capacity in HF patients. Testosterone replacement in these patients has been studied and shows an important therapeutic to enhance functional capacity and muscle strength. However it is not known the role of this medical treatment on muscle anabolic process as well as on body composition. Physical exercise as a non-medication treatment has been widely recommended to reduce MSNA, enhance peripheral blood flow, increase muscle strength and improve quality of life. However, the combination of the strategies of physical exercise associated with testosterone replacement therapy is not known in HF patients. Methods. 24 HF patients were randomized in 3 groups: Training (TR, n=9), Testosterone (T, n=8) and Training+Testosterone (TRT, n=7). MSNA was recorded by microneurography technic. Forearm blood flow was evaluated by venous occlusion plethysmography. Body composition was measured by densitometry (DEXA). Muscle biopsy was done in vastus lateralis to evaluate the cross-sectional area and type of fibers. Quality of life was assessed by Minnesota living with heart failure questionnaire. Aerobic exercise training on a bicycle was performed 3 times per week, with 40 minutes of exercise per session, for a period of 4 months. Testosterone replacement was performed by intramuscular administration of testosterone undecylate for a period of 4 months. Results. After 4 months testosterone levels were restored in all groups. MSNA decreased in TR and TRT groups. There was no increase in blood flow between groups. Oxygen consumption increased in all groups, but only the TRT group showed increase in maximum power to exercise. Lean body mass increased significantly only in the TRT group. We did not observe changes in bone mineral content between groups. Only TRT group significantly increased the cross-sectional area of type I fibers (oxidative). The quality of life improved only in TR and TRT groups. Conclusions. Exercise training associated with testosterone replacement therapy was more effective in reducing MSNA, increase functional capacity, muscle strength, lean mass with a significant increase in type I fibers. Our results emphasize the importance of physical exercise in patients with HF and bring a new perspective to association testosterone for patients with hypogonadism

Εισαγωγή: Το σύνδρομο της μεμονωμένης ανεπάρκειας της εκλυτικής ορμόνης των γοναδοτροπινών (IGD) χαρακτηρίζεται από μεμονωμένη λειτουργική ανεπάρκεια της υποθαλαμικής παραγωγής ή/και έκκρισης της GnRH οδηγώντας σε μεμονωμένη ανεπάρκεια των γοναδοτροπινών με φυσιολογική λειτουργικότητα των υπολοίπων υποφυσιακών ορμονών. Η συνύπαρξη IGD και ανοσμίας αναφέρεται ως σύνδρομο Kallmann (ΣΚ), ενώ η απουσία οσφρητικής διαταραχής αναφέρεται ως ιδιοπαθής υπογοναδοτροφικός υπογοναδισμός (ΙΥΥ). Σκοπός: Σκοπός της μελέτης είναι η περιγραφή των φαινοτυπικών χαρακτηριστικών ασθενών με μεμονωμένη ανεπάρκεια GnRH (ΙΥΥ και ΣΚ), η διερεύνηση της ύπαρξης μεταλλάξεων στα γονίδια KAL1, FGFR1 (υποδοχέας του αυξητικού παράγοντα των ινοβλαστών 1), PROK2 (προκινετισίνη 2), PROKR2 (υποδοχέας της προκινετισίνης 2) και GPR54 (KISS1R: υποδοχέας της κισσπεπτίνης) στους ασθενείς αυτούς, καθώς και η συσχέτιση μεταξύ του γονότυπου των ασθενών και ειδικών κλινικών φαινοτύπων. Επίσης διερευνείται η παρουσία μεταλλάξεων στο γονίδιο KAL1 σε ομάδα φαινομενικά υγιών παιδιών με ετερόπλευρη αγενεσία/δυσγενεσία νεφρού (ΕΝΑ), σε μια προσπάθεια καθορισμού της συχνότητας των μεταλλάξεων του γονιδίου KAL1 στην ΕΝΑ. Τέλος πραγματοποιείται in-vitro λειτουργικός έλεγχος δύο σημειακών μεταλλάξεων του γονιδίου FGFR1 που επηρεάζουν το ίδιο αμινοξύ της πρωτεΐνης (R254W και R254Q), με στόχο τη συσχέτιση των in-vitro ευρημάτων με τον κλινικό φαινότυπο των ασθενών. Ασθενείς: Μελετήθηκαν συνολικά εξήντα έξι (66) ασθενείς με μεμονωμένη ανεπάρκεια GnRH (26 με ΣΚ και 40 με ΙΥΥ), στους οποίους πραγματοποιήθηκε μοριακός έλεγχος των γονιδίων KAL1, FGFR1, PROK2, PROKR2 και GPR54. Επίσης μελετήθηκαν 13 παιδιά (ηλικίας κάτω των 15 ετών) στα οποία υπήρχε απεικονιστικά επιβεβαιωμένη συγγενής ετερόπλευρη νεφρική αγενεσία/δυσγενεσία, η οποία δεν παρατηρήθηκε στα πλαίσια γνωστού συνδρόμου και τα οποία ελέχθησαν για την παρουσία μεταλλάξεων στο γονίδιο KAL1. Μέθοδοι: Η μεθοδολογία του μοριακού γονιδιακού ελέγχου περιλάμβανε την απομόνωση DNA γονιδιώματος από δείγμα ολικού αίματος, τον εκλεκτικό πολλαπλασιασμό των εξονίων των υπό μελέτη γονιδίων με την αλυσιδωτή αντίδραση της πολυμεράσης (PCR amplification) και τον προσδιορισμό της αλληλουχίας του DNA στα προϊόντα της PCR (DNA sequencing). Ο in-vitro λειτουργικός έλεγχος των δύο μεταλλαγμένων μορφών του υποδοχέα FGFR1 (R254W και R254Q) περιλάμβανε την μελέτη της σηματοδοτικής δραστηριότητας του υποδοχέα κατόπιν διέγερσής του από τον προσδέτη FGF2, καθώς και τον προσδιορισμό των επιπέδων έκφρασης των μεταλλαγμένων μορφών του υποδοχέα FGFR1, τα οποία συγκρίθηκαν με τα αντίστοιχα του φυσιολογικού υποδοχέα (WT=wild type). Η μετάδοση σήματος του υποδοχέα FGFR1 αξιολογήθηκε με την τεχνική ανίχνευσης δραστηριότητας του γονιδίου αναφοράς της λουσιφεράσης. Η μέτρηση των επιπέδων της ολικής έκφρασης του FGFR1 πραγματοποιήθηκε με την τεχνική της ανάλυσης πρωτεϊνών με ηλεκτροφόρηση σε πήκτωμα πολυακριλαμιδίου, ακολουθούμενη από την τεχνική της ανάλυσης κατά Western. Η εκτίμηση της ενδοκυττάριας ωρίμανσης του πρωτεϊνικού μορίου του υποδοχέα FGFR1 έγινε μέσω ενζυμικής πέψης της γλυκοπρωτεΐνης με ενδογλυκοσιδάσες, ενώ ο υπολογισμός των επιπέδων έκφρασης του FGFR1 στην κυτταροπλασματική μεμβράνη έγινε με την πρόσδεση ραδιοσημασμένου αντισώματος (radiolabelled antibody binding assay). Αποτελέσματα: Εκ του μοριακού γονιδιακού ελέγχου που πραγματοποιήθηκε στους ασθενείς με IGD εντοπίσθηκαν πέντε διαφορετικές μεταλλάξεις στο γονίδιο KAL1 σε τρεις άρρενες ασθενείς με σύνδρομο Kallmann (Ε514Κ, Α660Τ, Ε37Κ, T235S, έλλειψη εξονίων 5-10), καθώς και μια σημειακή μετάλλαξη στο γονίδιο FGFR1 (R254W) σε έναν άρρενα ασθενή με ιδιοπαθή υπογοναδοτροφικό υπογοναδισμό. Εκ του in-vitro λειτουργικού ελέγχου των δύο σημειακών μεταλλάξεων του γονιδίου FGFR1 (R254W και R254Q) που μελετήθηκαν, προέκυψε ότι η μέγιστη σηματοδοτική δραστηριότητα για τη μεταλλαγμένη μορφή του υποδοχέα R254W παρουσιάζει μείωση κατά 45% σε σύγκριση με τον wild-type υποδοχέα (p<0.01), ενώ η μέγιστη απάντηση της μεταλλαγμένης μορφής R254Q μειώνεται κατά 15% σε σχέση με το wild-type υποδοχέα, διαφορά που δεν αναδείχθηκε στατιστικά σημαντική. Ωστόσο και οι δυο μεταλλαγμένες μορφές R254W και R254Q εμφανίζουν ελαττωμένα επίπεδα ολικής έκφρασης (40% και 30% μείωση σε σχέση με τον wild-type, αντίστοιχα), ενώ η πρωτεϊνική ωρίμανση δεν φαίνεται να επηρεάζεται. Τέλος η έκφραση των μεταλλαγμένων μορφών R254W και R254Q επί της κυτταρικής επιφάνειας παρουσιάζεται σημαντικά ελαττωμένη (35%, p<0.01 και 15%, p<0.05, αντιστοίχως). Εκ του μοριακού γονιδιακού ελέγχου που πραγματοποιήθηκε στους ασθενείς με ΕΝΑ βρέθηκε μετάλλαξη του KAL1 σε έναν ασθενή 12 ετών, ο οποίος εμφάνιζε συνοδό ανοσμία, συγκινησία άνω άκρων και κρυψορχία. Στον ασθενή αυτόν τέθηκε η γενετική διάγνωση του ΣΚ και αργότερα υποβλήθηκε σε έγκαιρη έναρξη θεραπευτικής αγωγής. Συμπεράσματα: Το ποσοστό των γνωστών μεταλλάξεων που έχουν εντοπισθεί στους ασθενείς με IGD του Ελλαδικού χώρου είναι πολύ μικρό και επομένως παραμένει πρόσφορο το πεδίο για περαιτέρω έρευνα προς την κατεύθυνση της διευκρίνησης της μοριακής αιτιοπαθογένειας της νόσου. Η σύγκριση φαινότυπου-γονότυπου των ασθενών με σύνδρομο Kallmann υποδεικνύει ότι η παρουσία συνοδού ετερόπλευρης αγενεσίας νεφρού αποτελεί ισχυρή ένδειξη για την ύπαρξη μεταλλαγών στο γονίδιο KAL1. Η ανεύρεση μεταλλάξεων του γονιδίου KAL1 σε παιδιά με ΕΝΑ έχει διττή σημασία· αφενός επιβεβαιώνει την εμπλοκή της ανοσμίνης-1 (του προϊόντος του γονιδίου KAL1) στην οργανογένεση του νεφρού και αφετέρου οδηγεί στην πρώιμη διάγνωση του ΣΚ. Ο μοριακός έλεγχος του γονιδίου KAL1 σε παιδιά με ΕΝΑ συστήνεται επί συνύπαρξης και άλλων κλινικών σημείων του ΣΚ (ανοσμία, κινήσεις καθρέπτη, κρυψορχία, μικροφαλλία) ή ανάδειξης οικογενειακού ιστορικού υπογοναδισμού και ανοσμίας. Ο συγκριτικός λειτουργικός έλεγχος δύο μεταλλάξεων του FGFR1 που επηρεάζουν το ίδιο αμινοξύ (R254W, R254Q) αναδεικνύει την απώλεια της λειτουργικότητας των μεταλλαγμένων μορφών του υποδοχέα in-vitro. Αν και από τον in-vitro λειτουργικό έλεγχο προκύπτει ότι η μετάλλαξη R254W είναι πιο σοβαρή από τη μετάλλαξη R254Q, ωστόσο δεν παρατηρείται συσχέτιση του βαθμού της απώλειας της in-vitro λειτουργικότητας των μεταλλαγμένων μορφών του υποδοχέα με τον κλινικό φαινότυπο των ασθενών που φέρουν αυτές τις μεταλλάξεις. / Background: Isolated GnRH deficiency (IGD) is characterized by a functional deficit of GnRH production or secretion in the hypothalamus resulting in the loss of pulsatile secretion of GnRH and in impaired gonadotropin release, in the setting of otherwise normal anterior pituitary anatomy and function and in the absence of secondary causes of hypogonadotropic hypogonadism (HH). Kallmann syndrome (KS) is characterized by the association of IGD and anosmia, whereas patients with normal olfactory function are referred as having normosmic Idiopathic Hypogonadotropic Hypogonadism (nIHH). Objective: The objective of the study was to describe the different patients phenotypes with IGD (KS and nIHH), to identify mutations in the KAL1, FGFR1, PROK2, PROKR2 and GPR54 genes and to correlate specific phenotypes with the patients genotypes. We also studied the presence of KAL1 mutations in young children with unilateral renal agenesis/dysgenesis, in order to determine the incidence of KAL1 gene mutations in this population. In addition, we attempted to define the in vitro functionality of two FGFR1 mutants (R254W and R254Q), resulting from two different amino acid substitutions of the same residue, and to correlate the in vitro findings to the patients phenotypes. Patients: A total of 66 patients with IGD (26 with KS and 40 with nIHH) were included in this study and mutation analysis of KAL1, FGFR1, PROK2, PROKR2 and GPR54 genes was performed for this group of patients. We also studied 13 children (up to the age of 15) with unilateral renal agenesis/dysgenesis, confirmed by imaging studies. Mutation analysis of KAL1 gene was performed for the later group of patients. Methods: Gene mutation analysis methodology included DNA extraction, polymerase chain reaction amplification, and DNA sequence analysis of all exons of the KAL1, FGFR1, PROK2, PROKR2 and GPR54 genes. The in-vitro functional studies of two FGFR1 mutants (R254W and R254Q) included evaluation of the mutant signaling activity and the expression levels, which were compared to the wild type (WT) receptor signaling activity and expression. Signaling activity was determined by a FGF2/FGFR1dependent transcription reporter assay. Receptor total expression levels were assessed by Western blot assay and receptor cell surface expression was measured by radiolabelled antibody binding assay. Results: We identified 5 different mutations in KAL1 gene in three unrelated male patients with KS (Ε514Κ, Α660Τ, Ε37Κ, T235S, deletion of exons 5-10 of KAL1 gene with STS gene deletion) and one point mutation in FGFR1 gene (R254W) in a male patient with nIHH. The in-vitro functional studies of the two FGFR1 mutants (R254W and R254Q) showed that R254W maximal receptor signaling capacity was reduced by 45% (p<0.01), while maximal signaling of R254Q was also reduced (−15%) but the reduction was not statistically significant relative to WT. However, both mutants displayed diminished total protein expression levels (40% and 30% reduction relative to WT, respectively), while protein maturation was unaffected. Accordingly, cell surface expression levels of the mutant receptors were also significantly reduced (35% p<0.01 and 15% p<0.05, respectively). Sequence analysis of KAL1 gene in the group of patients with unilateral renal agenesis/dysgenesis revealed genetic defects in KAL1 gene in a 12 year old child with associated anosmia, bimanual synkinesis of upper limbs and cryptorchidism. The genetic diagnosis of Kallmann syndrome was established in this case, enabling a prompt therapeutic intervention for puberty induction at a later stage. Conclusions: Up to date very few mutations have been described in patients with IGD in the Greek population and the genetic causes of IGD still remains unclear in the majority of cases, pointing out the importance of further studies for determining the molecular pathogenesis of the disease. The phenotype of renal agenesis/dysgenesis strongly indicates the existence of KAL1 gene defects in the genotype of patients with sporadic KS, providing evidence for the X-linked mode of inheritance and offering the opportunity for genetic counseling. The detection of KAL1 gene mutations in children with unilateral renal agenesis (URA) not only confirms the involvement of anosmin-1 (the product of KAL1 gene) in kidney organogenesis, but it can also lead to an early prepubertal diagnosis of KS. Sequence analysis of KAL1 gene in patients with URA is recommended in those cases with associated clinical signs of KS (e.g. anosmia, mirror movements, cryptorchidism, microphallus) or with familial history of hypogonadism or/and anosmia. The comparative functional analysis of two FGFR1 mutations affecting the same residue (R254W, R254Q) in two unrelated patients with IGD showed that both are loss-of-function mutations and that a tryptophan substitution at R254 (R254W) is more disruptive to receptor structure than the more conserved glutamine substitution (R254Q). However, no clear correlation between the severity of in vitro loss-of-function and phenotypic presentation could be assigned.

O mecanismo de controle da secreção de GnRH inclui diversas vias neuronais. Estudos em modelos animais identificaram genes que codificam fatores de transcrição, tais como TTF-1 (thyroid transcription factor 1) e EAP1 (enhanced at puberty), que atuam no controle transcricional de genes codificadores de fatores excitatórios (KiSS1 e GnRH) e inibitórios (preproencefalinas) regulando a secreção de GnRH. Em primatas, a expressão de EAP1 e TTF-1 aumenta, no início da puberdade, nas regiões hipotalâmicas envolvidas na secreção de GnRH. Nos modelos animais, a deleção pós-natal de TTF-1 e o silenciamento do EAP1 provocam atraso puberal e prejuízo na função reprodutiva. TTF-1 também está envolvido na morfogênese diencefálica, por meio da via de sinalização da família Sonic-Hedgehog. Anormalidades na secreção de GnRH resultam em distúrbios puberais, que variam de puberdade precoce central (PPC) a hipogonadismo hipogonadotrófico. Hipotetizamos que anormalidades genéticas no TTF-1 e EAP1 estejam envolvidas na patogênese dos distúrbios puberais centrais. A PPC pode ser idiopática ou devido a causas orgânicas, sendo o hamartoma hipotalâmico, uma malformação congênita não neoplásica, a mais conhecida. Os pacientes com PPC devido a hamartoma hipotalâmico podem cursar com alterações neurológicas e cognitivas. Nossos objetivos foram: estudar as regiões codificadora do TTF-1 e do EAP1 e a região promotora do TTF-1 em pacientes com distúrbios puberais centrais; estabelecer a prevalência, taxa de penetrância e modo de herança da forma familial de PPC e caracterizar as manifestações neurológicas e neurocognitivas de pacientes com PPC devido a hamartoma hipotalâmico. Foram selecionados 133 pacientes com distúrbios puberais centrais - PPC idiopática (n=71), PPC devido a hamartoma hipotalâmico (n=15) e hipogonadismo hipogonadotrópico isolado normósmico (HHIn) (n=47) - e controles (n=53). Os genes TTF-1 e EAP1 foram amplificados e submetidos a sequenciamento automático. Os tratos de poliglutamina e polialanina no EAP1 foram estudados por software de análise de tamanho de fragmento (GeneScan). A avaliação neurológica e neurocognitiva dos pacientes com PPC devido a hamartoma hipotalâmico consistiu de exame neurológico, eletroencefalograma, ressonância magnética de encéfalo e aplicação da escala de inteligência (WISC-III, WAIS-III, WPPSIR). Identificamos 25% de casos familiais de PPC, com modo de herança autossômica dominante e taxa de penetrância de 67,5%. Variantes alélicas no TTF-1 não foram identificadas nos pacientes estudados. No gene EAP1 foram identificadas quatro variantes alélicas sinônimas: p.E87E, p.A163A, p.Y415Y e uma nova variante alélica p.C758C, encontradas em pacientes com PPC e HHIn. A distribuição das frequências alélica e genotípica das variantes alélicas do EAP1 não diferiram entre pacientes com PPC, HHIn e controles (p >0,05). Nas regiões poliglutamina e polialanina 5 distal foi identificada variação similar no número de repetições glutamina e alanina em pacientes e controles. Não houve diferença significativa da frequência alélica em relação ao número de repetições glutamina e alanina entre os grupos PPC e HHIn (p >0,05). A avaliação neurológica dos pacientes com PPC devido a hamartoma hipotalâmico revelou epilepsia gelástica e crises focais com generalização em 3/15 (20%) pacientes. Não houve diferença significativa entre a mediana do maior diâmetro dos hamartomas dos pacientes com PPC com e sem epilepsia (13 e 10 mm, respectivamente). Quanto à forma, 10 hamartomas eram sésseis e 5 pedunculados, sendo que a forma pedunculada foi detectada exclusivamente em pacientes sem epilepsia. A avaliação neurocognitiva em 11 dos 15 pacientes com PPC devido a hamartoma hipotalâmico detectou 2 pacientes com epilepsia com QI significativamente menor que o grupo sem epilepsia (p <0,05). Em conclusão, (i) a considerável prevalência de casos familiais de PPC reforça a influência dos fatores genéticos na puberdade humana; (ii) mutações germinativas no TTF-1 e no EAP1 não estão envolvidas na patogênese dos distúrbios puberais centrais; (iii) a função neurocognitiva reduzida nos pacientes com hamartoma e epilepsia sugere um efeito deletério das crises convulsivas no sistema nervoso central / GnRH secretion control involves multiple neuronal pathways. Animal studies have identified genes which codifies transcription factors, such as TTF-1 (thyroid transcription factor 1) and EAP1 (enhanced at puberty), that act in the transcriptional control of genes that codifies excitatory (KiSS1 and GnRH) and inhibitory factors (preproenkephalines) regulating GnRH secretion. In nonhuman primates, expression of EAP1 and TTF-1 are increased at the hypothalamic regions involved in GnRH secretion, at the beginning of puberty. In animal models, post-natal TTF-1 deletion and silencing of EAP1 lead to pubertal delay and damage of reproductive function. TTF-1 is also involved in diencephalic morphogenesis, through signalization via Sonic-Hedgehog family. Abnormalities in GnRH secretion are responsible for pubertal disorders, varying from central precocious puberty (CPP) to hypogonadotropic hypogonadism. We hypothesized that genetic anomalies at TTF-1 and EAP1 are involved in the pathogenesis of central pubertal disorders. CPP may be idiopathic or due to organic alterations and hypothalamic hamartoma, a non-neoplasic congenital malformation, is the most frequent known organic cause. Patients with CPP due to hypothalamic hamartoma may have neurological and cognitive disfunctions. Our aims were: to evaluated the codifying region of TTF-1 and EAP1 and the promoter region of TTF-1 in patients with central pubertal disorders; to establish the prevalence, penetrance rate and inheritance mode of familial CPP and to characterize neurologic and neurocognitive aspects of patients with CPP due to hypothalamic hamartoma. We selected 133 patients with central pubertal disorders idiopathic CPP (n=71), CPP due to hypothalamic hamartoma (n=15) and normosmic isolated hypogonadropic hypogonadism (nIHH) (n=47) - and controls (n=53). TTF-1 and EAP1 genes were amplified and sequenced. Polyglutamine and polyalanine tracts of EAP1 were studied by a fragment size analyser software (GeneScan). Neurologic and neurocognitive evaluation of CPP patients due to hypothalamic hamartoma consisted of neurologic exam, electroencephalogram, brain magnetic resonance and application of intelligence scale (WISC-III, WAIS-III, WPPSI-R). We identified 25% of familial CPP cases with autosomal dominant mode of inheritance and penetrance rate of 67.5%. No TTF-1 allelic variants were identified in the patients analysed. At EAP1 gene, four synonimous allelic variants were identified: p.E87E, p.A163A, p.Y415Y and a new allelic variant p.C758C, found in CPP and nIHH patients. The allelic and genotypic distribution of theses variants of EAP1 did not differ among patients with CPP and nIHH, and controls (p >0.05). At polyglutamine and 5 distal polyalanine region, similar glutamine and alanine repeats variation was found. No significative difference of allelic frequency distribution regarding the number of glutamines and alanines repeats was found among the studied groups (p >0.05). Neurologic evaluation of CPP patients due to hypothalamic hamartoma revealed epilepsy and focal crisis with generalization in 3/15 (20%) of the patients. No significant difference between the median of the larger diameter of hypothalamic hamartoma of CPP patients with and without epilepsy was found (10 mm and 13 mm, respectively). Regarding the shape, 10 hamartomas were sessile and 5 pedunculated, and the pedunculated shape was found only in non epileptic patients. Neurocognitive evaluation performed in 11 of the 15 patients with CPP due to hypothalamic hamartoma detected 2 patients with epilepsy whose IQ were significantly lower than the IQ found in the group without epilepsy (p <0.05). In conclusion, (i) the considerable prevalence of familial CPP cases reinforce the influence of genetic factors in human puberty; (ii) germinative mutations in TTF-1 and EAP1 are not involved in the pathogenesis of central pubertal disorders; (iii) reduced neurocognitive function in patients with hypothalamic hamartoma and epilepsy suggests a deleterious effect of crisis at the central nervous system

A resposta do LH e do FSH ao estímulo com GnRH, realizado em estádio pré-puberal em pacientes com hipopituitarismo acompanhados até a idade puberal, são úteis para predizer o diagnóstico da deficiência de gonadotrofinas, principalmente nas meninas. O estudo da região codificadora do gene LH em pacientes com hipogonadismo hipogonadotrófico e concentrações normais de LH revelou 5 variantes alélicas. A freqüência das variantes alélicas Arg8 e Thr15 foi similar entre hipogonádicos e adultos normais e a sua presença não interferiu nas concentrações séricas do LH. O estudo das isoformas do LH mostrou um predomínio das isoformas ácidas do LH em hipogonádicos e indivíduos normais, não permitindo atribuir à sua presença a baixa atividade biológica do LH imunorreativo encontrado em 13% dos hipogonádicos / LH and FSH responses to GnRH stimulation carried out in the pre-pubertal stage in patients with hypopituitarism followed until the pubertal stage are useful tools for predicting the gonadotropin deficiency diagnosis, especially in girls. The study of the codifying region of the LH gene in patients with hypogonadotropic hypogonadism and normal LH levels disclosed 5 allelic variants. The frequencies of the allelic variants Arg8 and Thr15 were similar between hypogonadic and normal adults, and their presence did not alter serum LH levels. The study of LH isoforms showed a predominance of acid LH isoforms in hypogonadic and normal subjects, which does not allow us to ascribe to their presence the low biological activity of the immunoreactive LH, found in 13% of the hypogonadic individuals

O sistema da procineticina desempenha um papel importante na migração dos neurônios secretores de GnRH e na neurogênese do bulbo olfatório. Camundongos com ablação dos genes que codificam a procineticina 2 (PROK2) e seu receptor (PROKR2) apresentaram fenótipos semelhantes ao da síndrome de Kallmann descrita em humanos. Mutações inativadoras nos genes PROK2 e PROKR2 foram identificadas em pacientes com hipogonadismo hipogonadotrófico isolado. Com base nestes achados, investigamos a presença de alterações estruturais nos genes PROK2 e PROKR2 em 107 pacientes brasileiros (63 com síndrome de Kallmann e 47 com hipogonadismo hipogonadotrófico isolado normósmico). Cem indivíduos brasileiros que relataram desenvolvimento puberal normal foram utilizados como grupo controle. As regiões codificadoras dos genes PROK2 e PROKR2 foram amplificadas utilizando-se oligonucleotídeos intrônicos específicos, seguida de purificação enzimática e sequenciamento automático. Duas mutações no gene PROK2 foram identificadas: a mutação p.G100fsX121 em homozigose presente em dois irmãos com síndrome de Kallmann; e a mutação p.I55fsX56 em heterozigose identiificada em um paciente com HHIn. Quatro mutações foram identificadas no gene PROKR2 (p.R80C, p.Y140X, p.L173R e p.R268C) em cinco pacientes com síndrome de Kallmann e um paciente com HHIn. Essas mutações não foram encontradas no grupo controle. As mutações do tipo missense, p.R80C, p.L173R e p.R268C foram identificadas em heterozigose. Mutações nos genes FGFR1, GnRHR, KiSS-1 e GPR54 foram excluídas nesses pacientes. O paciente portador da mutação p.R268C do PROKR2 apresentou deleção dos exons 1 e 2 do gene KAL1. Adicionalmente, as mutações p.R80C e p.R268C foram identificadas em heterozigose em parentes de primeiro grau assintomáticos dos casos índices. A nova mutação p.Y140X do PROKR2, única alteração em homozigose, foi identificada em um paciente com micropênis, criptorquidia bilateral, anosmia e palato ogival. Os pais deste paciente eram portadores da mutação p.Y140X em heterozigose e relataram desenvolvimento puberal normal e ausência de anormalidades olfatórias. Estudos in vitro da nova mutação p.R80C localizada na primeira alça intracelular demonstraram que o acúmulo de fofatidil-inositol (IP), assim como a ativação da via da MAPK foram significativamente afetadas em células transfectadas com o receptor mutado em relação ao receptor selvagem, indicando que a mutação p.R80C determina uma menor atividade do receptor. Avaliação da expressão por Western blot mostrou uma diminuição na expressão do receptor mutado R80C e uma maior expressão de receptores imaturos. Esses achados sugeriram o papel crítico da arginina localizada na posição 80 na atividade normal do receptor. Em conclusão, expandimos o repertório de mutações deletérias nos genes PROK2 e PROKR2 em pacientes com hipogonadismo hipogonadotrófico isolado. A haploinsuficiência do PROKR2 não foi suficiente para causar síndrome de Kallmann ou HHIn, entretanto mutações inativadoras em homozigose nos genes PROK2 e PROKR2 foram responsáveis pelo fenótipo reprodutivo e olfatório anormal, em concordância com os estudos prévios de ablação gênica em modelos animais. Arginina localizada na posição 80 do PROKR2 desempenha um papel crucial na adequada maturação do receptor / Physiological activation of the prokineticin pathway has a critical role in olfactory bulb morphogenesis and GnRH secretion. Knock-out mice for genes that encode prokineticin 2 (PROK2) and the prokineticin receptor 2 (PROKR2) exhibited a phenotype similar to the Kallmann syndrome (KS). Inactivating mutations in PROK2 and PROKR2 have been identified in patients with isolated hypogonadotropic hypogonadism. Based on these findings, we investigated the presence of inactivating mutations of the genes PROK2 and PROKR2 in Brazilian patients with isolated hypogonadotropic hypogonadism associated or not with olfactory abnormalities and performed in vitro studies of the new identified mutations. We studied 107 patients with HH (63 with Kallmann syndrome and 44 with normosmic HH) and 100 control individuals. The coding regions of PROK2 and PROKR2 were amplified by polymerase chain reaction followed by enzymatic purification and direct automatic sequencing. In PROK2, two known frameshift mutations were identified. Two brothers with Kallmann syndrome harbored the homozygous p.G100fsX121 mutation, whereas one male with normosmic HH harbored the heterozygous p.I55fsX56 mutation. In PROKR2, four distinct mutations (p.R80C, p.Y140X, p.L173R and p.R268C) were identified in five patients with Kallmann syndrome and in one patient with normosmic HH. These mutations were not found in the control group. The p.R80C and p.R268C missense mutations were identified in heterozygous state in the HH patients and in their asymptomatic first-degree relatives. The p.L173R was also identified in heterozygous state. In addition, no mutations of FGFR1, GnRHR, KiSS-1 or GPR54 were identified in these patients. The patient with the PROKR2 mutation p.R268C also has a deletion of the exon 1 and 2 in the gene KAL1. Notably, the new nonsense mutation (p.Y140X) was identified in homozygous state in an anosmic boy with micropenis, bilateral cryptorchidism and high-arched palate. His asymptomatic parents were heterozygous for this severe defect. In vitro studies of the new mutation, p.R80C, were performed in order to access the mechanism by which this mutation could affect the activity of the PROKR2. In vitro studies showed that the amount of fofatidil-inositol (PI) and the activation of MAPK were significantly lower in cells transfected with the R80C mutant receptor than in cells transfected with the wild receptor, indicating that this variant is a loss-of-function mutation. Binding studies and Western blot showed a reduction in the expression levels of the receptor in the plasma membrane and in whole cell, respectively. Additionally, Western blot analysis of R80C PROKR2 revealed an additional smaller molecular weight band that represents the presence of immature unglycosylated receptors. The arginine 80 in ICL1 is important for post-translational processing of PROKR2. In conclusion, we expanded the repertoire of PROK2 and PROKR2 mutations in patients with HH and showed that PROKR2 haploinsufficiency is not sufficient to cause Kallmann syndrome or normosmic HH, whereas homozygous loss-of-function mutations either in PROK2 or PROKR2 are sufficient to cause disease phenotype, in accordance with the Prokr2 and Prok2 knockout mouse models. In vitro studies suggested that the arginine located at position 80 of the receptor seems to play an important role in the receptor function

A kisspeptina, codificada pelo gene KISS1, é um neuropeptídeo crucial na regulação do início da puberdade. A kisspeptina estimula a secreção hipotalâmica do hormônio liberador de gonadotrofinas (GnRH) após se ligar ao seu receptor GPR54. Mutações inativadoras do GPR54 são atualmente consideradas como uma causa rara de hipogonadismo hipogonadotrófico isolado (HHI) normósmico. Recentemente, uma mutação ativadora no receptor GPR54 foi implicada na patogênese da puberdade precoce dependente de gonadotrofinas (PPDG). Com base nesses achados, levantamos a hipótese de que alterações no gene KISS1 poderiam contribuir para a patogênese de distúrbios puberais centrais. O objetivo do presente estudo foi investigar a presença de variantes no gene KISS1 em pacientes com PPDG e HHI. Sessenta e sete crianças brasileiras com PPDG (63 meninas e 4 meninos) e 61 pacientes com HHI (40 homens e 21 mulheres) foram selecionados, incluindo casos esporádicos e familiares em ambos os grupos. A população controle consistiu de 200 indivíduos com história de desenvolvimento puberal normal. A região promotora e os 3 exons do gene KISS1 foram amplificados e submetidos a sequenciamento automático. Duas novas variantes no gene KISS1, p.P74S e p.H90D, foram identificadas em duas crianças não relacionadas, portadoras de PPDG idiopática. Ambas as variantes estão localizadas na região amino-terminal da kisspeptina-54 e estavam ausentes em 400 alelos controles. A variante p.P74S foi identificada em heterozigose em um menino que desenvolveu puberdade com um ano de idade. Sua mãe e avó materna, que apresentavam história de desenvolvimento puberal normal, eram portadoras da mesma variante em heterozigose, sugerindo penetrância incompleta e/ou herança sexo-dependente. A variante p.H90D foi identificada em homozigose em uma menina com PPDG, que desenvolveu puberdade aos seis anos de idade. Sua mãe, com história de menarca aos dez anos de idade, era portadora da mesma variante em heterozigose. Células transfectadas estavelmente com GPR54 foram estimuladas com concentrações crescentes de kisspeptina-54 (kp-54) humana selvagem ou contendo as mutações (kp-54 H90D e kp-54 P74S) e o acúmulo de fosfato de inositol (IP) foi medido. Nos estudos in vitro, a kp-54 P74S apresentou uma capacidade de ativação do receptor GPR54 semelhante à kp-54 selvagem. A kp-54 p.H90D mostrou uma ativação da sinalização do receptor significativamente mais potente que a kp-54 selvagem, sugerindo que essa é uma mutação ativadora. No grupo de HHI, uma nova variante (c.588-589insT) foi identificada em heterozigose na região 3 não traduzida do gene KISS1 em um paciente do sexo masculino. O papel dessa variante no fenótipo de HHI permanece indeterminado. Em conclusão, duas mutações no gene KiSS1 foram descritas pela primeira vez em associação com PPDG. / Kisspeptin, encoded by the KISS1 gene, is an important regulator of puberty onset. After binding to its receptor GPR54, kisspeptin stimulates gonadotropin-releasing hormone secretion by the hypothalamic neurons. Inactivating GPR54 mutations are a rare cause of normosmic isolated hypogonadotropic hypogonadism (IHH). Recently, a unique GPR54 activating mutation was implicated in the pathogenesis of gonadotropin dependent precocious puberty (GDPP). Based on these observations, we hypothesized that mutations in the KISS1 gene might be associated with central pubertal disorders. The aim of this study was to investigate KISS1 mutations in idiopathic GDPP and normosmic IHH. Sixty-seven Brazilian children (63 girls and 4 boys) with idiopathic GDPP and 61 patients with normosmic IHH (40 men and 21 women) were selected. Familial and sporadic cases were included in both groups. The control population consisted of 200 individuals who had normal timing of puberty. The promoter region and the 3 exons of the KISS1 gene were amplified and automatically sequenced. Two novel KISS1 missense mutations, p.P74S and p.H90D, were identified in two unrelated children with idiopathic GDPP. Both mutations were absent in 400 control alleles and are located in the amino-terminal region of kisspeptin-54. The p.P74S mutation was identified in the heterozygous state in a boy who developed puberty at 1 yr of age. His mother and maternal grandmother, who had normal pubertal development, were also heterozygous for the p.P74S mutation, suggesting incomplete penetrance and/or sex-dependent inheritance. The p.H90D mutation was identified in the homozygous state in a girl with GDPP, who developed puberty at 6 yr of age. Her mother, who had menarche at 10 yr of age, carried the p.H90D mutation in the heterozygous state. CHO cells stably transfected with GPR54 were stimulated with different concentrations of synthetic human wild type or mutant kisspeptin-54 (KP54) and inositol phosphate (IP) accumulation was measured. In vitro studies revealed that the capacity of the p.P74S mutant KP54 to stimulate IP production was similar to the wild type. The p.H90D kisspeptin-54 showed a significantly more potent activation of GPR54 signaling in comparison to the wild type in vitro, suggesting a gain-of-function mutation. In the IHH group, a heterozygous variant in the 3 UTR of the KISS1 gene (c.588-589insT) was identified. The role of this variant in the IHH phenotype remains to be determined. In conclusion, two KiSS1 mutations were described for the first time in association with GDPP.