A Síndrome de Kallmann (SK), associação entre hipogonadismo hipogonadotrófico e distúrbio olfatório (hiposmia ou anosmia), é causada por uma deficiência da migração neuronal que envolve as células produtoras do hormônio liberador de gonadotrofinas e os neurônios olfatórios, com origem embriológica comum. O primeiro gene descrito, KAL1, codifica uma proteína chamada anosmina, que possui homologia com moléculas de adesão axonal envolvidas na migração neuronal. Dentre as anormalidades fenotípicas descritas na SK, destacam-se a malformação das estruturas do rinencéfalo (bulbos e sulcos olfatórios) e a presença, em parte dos pacientes, de uma alteração neurológica específica, os movimentos em espelho (ME). No presente trabalho estudamos 21 pacientes com SK, comparando com um grupo controle (n=16), utilizando técnicas qualitativas e quantitativas de imagem por Ressonância Magnética (RM), com os objetivos de (I) correlacionar as diferentes alterações radiológicas do encéfalo com os achados clínicos, laboratoriais e a presença de mutações gênicas; (II) caracterizar qualitativa e quantitativamente as alterações do rinencéfalo; e (III) investigar possíveis alterações associadas aos ME, dando atenção às duas principais hipóteses para sua causa, desenvolvimento anormal do trato corticoespinhal e deficiência do mecanismo inibitório contra-lateral via corpo caloso. Para estudo do rinencéfalo utilizamos especialmente imagens coronais ponderadas em T2 com cortes finos, avaliando subjetivamente e posteriormente medindo o volume dos bulbos olfatórios, comprimento e profundidade dos sulcos. Para estudo dos ME utilizamos a técnica de morfometria baseada em voxel (VBM), procurando alterações volumétricas da substancia branca (SB) e cinzenta (SC), seguida da avaliação de alterações da SB com as técnicas de relaxometria (RL) e cálculo da taxa de transferência de magnetização (TTM). Dezoito (85%) pacientes apresentaram graus variáveis de acometimento das estruturas olfatórias. Demonstramos que a aplasia dos bulbos e/ou sulcos olfatórios foi o achado mais comum na SK, e a presença de aplasia de bulbo olfatório teve excelente concordância com a presença de anosmia no teste olfativo. O VBM otimizado mostrou áreas alteradas da SC envolvendo o córtex motor de maneira diferente nos pacientes com e sem ME, compatíveis com uma resposta cortical hipertrófica à uma decussação deficiente do trato corticoespinhal nos pacientes com SK e ME. Ainda, as alterações da SC nos pacientes sem ME podem representar mecanismos mais complexos determinando a presença ou não do sinal. Apesar do nosso estudo de VBM não mostrar alteração significativa de volume da SB, as avaliações com RL e TTM mostraram áreas de alteração de sinal, demonstrando a presença de desmielinização e/ou desorganização axonal na SB dos pacientes com SK, envolvendo diferentemente o sistema motor dos pacientes com e sem ME, sendo compatível com os estudos prévios que sugerem a associação entre uma anomalia do trato corticoespinhal e uma deficiência no mecanismo inibitório inter-hemisférico como responsáveis pela persistência patológica dos ME. A análise da TTM demonstrou ainda alteração em topografia da decussação das pirâmides bulbares que pode representar o desarranjo primário desta região, com as alterações das fibras a montante, superiormente (evidenciadas na RL e TTM), e do córtex motor (evidenciadas no VBM), possivelmente sendo secundárias e acometendo diferentemente os pacientes com e sem ME. / Kallmann syndrome (KS) is defined by the association of hypogonadotropic hypogonadism with olfaction disturbance (hyposmia or anosmia). It is caused by a neuronal migration arrest that involves both the gonadotropin releasing hormone (GnRH) and the olfactory neurons, which have a common embryonic origin. The first gene described, KAL1, encodes a protein named anosmin, which shows a strong homology to axonal adhesion molecules involved in neuronal migration and axonal pathfinding. Various phenotypic abnormalities have been described in KS, including olfactory bulbs and sulci aplasia or hypoplasia and specific neurologic disorders, such as mirror movements (MM). In this study we evaluated 21 patients with KS, comparing with a control group (n=16), using qualitative and quantitative techniques with Magnetic Resonance Imaging (MRI), with the following purposes: (I) correlate the brains radiologic alterations with the clinical, laboratorial and genetic findings; (II) characterize the rhinencephalon alterations; and (III) investigate MM etiology, addressing the two main hypotheses concerning its cause, abnormal development of the primary motor system, involving the ipsilateral corticospinal tract, and lack of contralateral motor cortex inhibitory mechanisms, mainly through the corpus callosum. For rhinencephalon evaluation, we specially used thin-section coronal T2-weighed images, which were reviewed and then objectively evaluated with the measurements of the olfactory bulbs and sulci. To study MM we used the voxel-based morphometry (VBM), to determine white (WM) and gray matter (GM) volume changes, and T2 relaxometry (T2R) and magnetization transfer ratio (MTR), searching for signal intensity changes in the WM. Eighteen (85%) patients presented different degrees of olfactory structures abnormalities, with the bulbs and/or sulci aplasia being the most common finding, and presence olfactory bulb aplasia showed excellent agreement with anosmia as determined by the smell identification clinical test. The optimized VBM study did not show significant white matter changes in patients with KS but showed gray matter alterations in keeping with a hypertrophic response to a deficient pyramidal decussation in patients with MM. In addition, gray matter alterations were observed in patients without MM, which can represent more complex mechanisms determining the presence or absence of this symptom. Even if the VBM did not show significant volume changes in WM, the evaluation with the T2R and MTR showed WM signal intensity alterations, differently involving patients with and without MM, in keeping with demyelinization and/or axonal disorder, in accordance with the involvement of a corticospinal tract anomaly and a deficient inhibitory interhemispheric mechanism in the etiology of MM. The MTR analysis also showed a different alteration in the pyramidal decussation, which can represent a primary disorder in this region, with all other alterations in the superior WM fibers and motor cortex possibly being secondary to this disarrangement, and involving patients with and without MM in a different manner.

A Síndrome de Kallmann (SK), associação entre hipogonadismo hipogonadotrófico e distúrbio olfatório (hiposmia ou anosmia), é causada por uma deficiência da migração neuronal que envolve as células produtoras do hormônio liberador de gonadotrofinas e os neurônios olfatórios, com origem embriológica comum. O primeiro gene descrito, KAL1, codifica uma proteína chamada anosmina, que possui homologia com moléculas de adesão axonal envolvidas na migração neuronal. Dentre as anormalidades fenotípicas descritas na SK, destacam-se a malformação das estruturas do rinencéfalo (bulbos e sulcos olfatórios) e a presença, em parte dos pacientes, de uma alteração neurológica específica, os movimentos em espelho (ME). No presente trabalho estudamos 21 pacientes com SK, comparando com um grupo controle (n=16), utilizando técnicas qualitativas e quantitativas de imagem por Ressonância Magnética (RM), com os objetivos de (I) correlacionar as diferentes alterações radiológicas do encéfalo com os achados clínicos, laboratoriais e a presença de mutações gênicas; (II) caracterizar qualitativa e quantitativamente as alterações do rinencéfalo; e (III) investigar possíveis alterações associadas aos ME, dando atenção às duas principais hipóteses para sua causa, desenvolvimento anormal do trato corticoespinhal e deficiência do mecanismo inibitório contra-lateral via corpo caloso. Para estudo do rinencéfalo utilizamos especialmente imagens coronais ponderadas em T2 com cortes finos, avaliando subjetivamente e posteriormente medindo o volume dos bulbos olfatórios, comprimento e profundidade dos sulcos. Para estudo dos ME utilizamos a técnica de morfometria baseada em voxel (VBM), procurando alterações volumétricas da substancia branca (SB) e cinzenta (SC), seguida da avaliação de alterações da SB com as técnicas de relaxometria (RL) e cálculo da taxa de transferência de magnetização (TTM). Dezoito (85%) pacientes apresentaram graus variáveis de acometimento das estruturas olfatórias. Demonstramos que a aplasia dos bulbos e/ou sulcos olfatórios foi o achado mais comum na SK, e a presença de aplasia de bulbo olfatório teve excelente concordância com a presença de anosmia no teste olfativo. O VBM otimizado mostrou áreas alteradas da SC envolvendo o córtex motor de maneira diferente nos pacientes com e sem ME, compatíveis com uma resposta cortical hipertrófica à uma decussação deficiente do trato corticoespinhal nos pacientes com SK e ME. Ainda, as alterações da SC nos pacientes sem ME podem representar mecanismos mais complexos determinando a presença ou não do sinal. Apesar do nosso estudo de VBM não mostrar alteração significativa de volume da SB, as avaliações com RL e TTM mostraram áreas de alteração de sinal, demonstrando a presença de desmielinização e/ou desorganização axonal na SB dos pacientes com SK, envolvendo diferentemente o sistema motor dos pacientes com e sem ME, sendo compatível com os estudos prévios que sugerem a associação entre uma anomalia do trato corticoespinhal e uma deficiência no mecanismo inibitório inter-hemisférico como responsáveis pela persistência patológica dos ME. A análise da TTM demonstrou ainda alteração em topografia da decussação das pirâmides bulbares que pode representar o desarranjo primário desta região, com as alterações das fibras a montante, superiormente (evidenciadas na RL e TTM), e do córtex motor (evidenciadas no VBM), possivelmente sendo secundárias e acometendo diferentemente os pacientes com e sem ME. / Kallmann syndrome (KS) is defined by the association of hypogonadotropic hypogonadism with olfaction disturbance (hyposmia or anosmia). It is caused by a neuronal migration arrest that involves both the gonadotropin releasing hormone (GnRH) and the olfactory neurons, which have a common embryonic origin. The first gene described, KAL1, encodes a protein named anosmin, which shows a strong homology to axonal adhesion molecules involved in neuronal migration and axonal pathfinding. Various phenotypic abnormalities have been described in KS, including olfactory bulbs and sulci aplasia or hypoplasia and specific neurologic disorders, such as mirror movements (MM). In this study we evaluated 21 patients with KS, comparing with a control group (n=16), using qualitative and quantitative techniques with Magnetic Resonance Imaging (MRI), with the following purposes: (I) correlate the brains radiologic alterations with the clinical, laboratorial and genetic findings; (II) characterize the rhinencephalon alterations; and (III) investigate MM etiology, addressing the two main hypotheses concerning its cause, abnormal development of the primary motor system, involving the ipsilateral corticospinal tract, and lack of contralateral motor cortex inhibitory mechanisms, mainly through the corpus callosum. For rhinencephalon evaluation, we specially used thin-section coronal T2-weighed images, which were reviewed and then objectively evaluated with the measurements of the olfactory bulbs and sulci. To study MM we used the voxel-based morphometry (VBM), to determine white (WM) and gray matter (GM) volume changes, and T2 relaxometry (T2R) and magnetization transfer ratio (MTR), searching for signal intensity changes in the WM. Eighteen (85%) patients presented different degrees of olfactory structures abnormalities, with the bulbs and/or sulci aplasia being the most common finding, and presence olfactory bulb aplasia showed excellent agreement with anosmia as determined by the smell identification clinical test. The optimized VBM study did not show significant white matter changes in patients with KS but showed gray matter alterations in keeping with a hypertrophic response to a deficient pyramidal decussation in patients with MM. In addition, gray matter alterations were observed in patients without MM, which can represent more complex mechanisms determining the presence or absence of this symptom. Even if the VBM did not show significant volume changes in WM, the evaluation with the T2R and MTR showed WM signal intensity alterations, differently involving patients with and without MM, in keeping with demyelinization and/or axonal disorder, in accordance with the involvement of a corticospinal tract anomaly and a deficient inhibitory interhemispheric mechanism in the etiology of MM. The MTR analysis also showed a different alteration in the pyramidal decussation, which can represent a primary disorder in this region, with all other alterations in the superior WM fibers and motor cortex possibly being secondary to this disarrangement, and involving patients with and without MM in a different manner.

No description available.

BACKGROUND: Pleiotropy is a biological phenomenon of a single gene exhibiting influence over several different seemingly disparate phenotypes. This phenomenon poses significant challenges to fully understanding the etiologies of many different Mendelian diseases. Two such Mendelian diseases are Idiopathic Hypogonadotropic Hypogonadism (IHH) and Craniosynostosis (CS). IHH results from the failure of differentiation, migration, secretion, or action of the GnRH neurons resulting in absent puberty and infertility. CS is characterized by premature fusion of one or more of the cranial sutures resulting in dysmorphic shape of the skull that can lead to life-threatening raised intercranial pressure requiring surgical intervention. Thus far, 77 genes have been implicated in IHH and 128 genes have been implicated CS, both representing ~50% of the cases in their respective diseases. Recent research has suggested a shared molecular landscape in CS and IHH but the full ensemble of this overlap is not known. OBJECTIVE: This study will attempt to utilize human genetics, bioinformatics, statistics, phenotype data of IHH patients, and the prior literature in order to ascertain the full extent of the shared biology of IHH and CS. METHODS: The gene sets of both IHH and CS were used in gene overlap statistical analysis to investigate shared genetics. Whole exome sequencing data from 1,395 patients from the IHH cohort of the Massachusetts General Hospital were used for gene-variant burden analysis to determine genetic overlap with CS. Detailed physician notes from this cohort were used to determine phenotypic presence of CS in IHH. Conversely, evidence of reproductive phenotypes in genetically characterized CS patients was gathered from the reported CS gene literature. The CS and IHH gene sets were also bioinformatically analyzed using both the Metascape and DAVID bioinformatic platforms for pathway annotation, protein-protein interaction (PPI), and functional interactions to provide evidence for the mechanism of shared biology. RESULTS: Of the 128 CS genes and 77 IHH genes, 4 were determined to be causal for both diseases with a further 3 considered as potentially causal candidates for both diseases. The 4 overlapping causal genes were tested using three different methods and this overlap was determined to be of statistical significance (p<0.05). Furthermore, the phenotypic review revealed that while there was not a significant enrichment for CS phenotypes in the IHH cohort, the literature review yielded 49 of 128 CS genes that were reported with phenotypic evidence of failure of the hypothalamic-pituitary portion of the HPG axis. Gene-variant burden analysis yielded nominal (p<0.05) enrichment in the IHH cohort for 17 CS genes, of which 3 were significant after Bonferroni multiple testing correction (p<0.00039). The CS/IHH gene sets were both enriched in 44 shared pathways according to Metascape and 17 shared pathways according to DAVID. PPI analysis yielded 3 shared communities between the two disorders with enrichment in fibroblast signaling, ossification, and cardiac chamber development. CONCLUSIONS: The shared biology between IHH and CS was significantly greater than what was previously appreciated. Shared pathways of the two gene sets point toward the neural crest origin of subpopulations of the GnRH neuron and cranial suture osteoblast as a possible foundation for this shared biology, as well as the migratory nature of these two cells and the role that many genes in both gene sets play in cellular motility. Several CS genes emerge as candidates for IHH and must be individually evaluated. Functional studies should be used to confirm and further unravel the underlying mechanisms for the biological overlap between these two diseases. This study may provide a model for preemptive in silico work prior to more expensive in vitro or in vivo studies of pleiotropy.

A síndrome de Kallmann (SK) é um distúrbio raro, caracterizado pela presença do Hipogonadismo Hipogonadotrófico Idiopático (HHI) associado à anosmia ou hiposmia (ausência ou déficit no sentido do olfato, respectivamente). O HHI tem como principal causa a deficiência do hormônio liberador de gonadotrofina (GnRH), com heterogeneidade genética significativa. Clinicamente é caracterizada por níveis plasmáticos baixos dos hormônios luteinizante (LH) e folículo-estimulante (FSH) associados com baixas concentrações de esteroides sexuais. O estabelecimento de um fenótipo cognitivo claro da SK pode ajudar na compreensão sobre o desenvolvimento das funções cognitivas humanas e a informar para um melhor manejo do paciente, com um acompanhamento mais apropriado. Neste estudo foi realizada uma bateria de avaliação neuropsicológica, caracterizando aspectos referentes aos quocientes intelectuais, atenção, funções executivas, memória, linguagem e habilidades visuoespaciais. Como resultados, o grupo de participantes diagnosticado de SK apresentou desempenho inferior em todas as áreas avaliadas, quando comparado ao grupo controle. Todavia, seu desempenho de maneira geral esteve dentro das faixas média e média-inferior, caracterizando assim um déficit leve das funções neuropsicológicas avaliadas. A memória declarativa de longa duração, no entanto, no grupo clínico esteve bastante prejudicada, com classificação dentro do espectro deficitário. Este resultado, aliado ao desempenho inferior em atividades que exigiam um julgamento de origem temporal e ao comprometimento da aprendizagem de associações condicionais arbitrárias (entre estímulos e respostas), sugere que a maior característica cognitiva presente no grupo clínico está na dificuldade de reter e selecionar informações apropriadas dentre os diversos estímulos e não apenas na recuperação destas informações. / The Kallmann syndrome (KS) is a rare disorder characterized by the presence of hypogonadotropic hypogonadism Idiopathic (IHH) associated with anosmia or hyposmia (absence or deficit in the sense of smell, respectively). The HHI\'s main causes deficiency of gonadotropin-releasing hormone (GnRH), with significant genetic heterogeneity. Clinically, it is characterized by low plasma levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH) associated with low concentrations of sex steroids. The establishment of a clear cognitive phenotype of SK can help in understanding the development of human cognitive functions and to report to a better management of the patient with a more appropriate accompaniment. In this study a battery of neuropsychological evaluation was performed, featuring aspects of intellectual quotients, attention, executive functions, memory, language and visuospatial skills. As a result, the diagnosed group of participants of KS showed lower performance in all areas evaluated, when compared to the control group. However, its performance in general was in the middle and lower-middle ranges, characterizing a slight deficit of assessed neuropsychological functions. The declarative memory of long term, however, in the clinical group was significantly impaired classified within the spectrum deficit. This result, coupled with lower performance in activities requiring a judgment of temporal origin and impairment of learning arbitrary conditional associations (between stimuli and responses), suggests that higher cognitive feature present in the clinical group is the difficulty of retaining and select information appropriate from the various stimuli, not just the recovery of this information.

A síndrome de Kallmann (SK) é um distúrbio raro, caracterizado pela presença do Hipogonadismo Hipogonadotrófico Idiopático (HHI) associado à anosmia ou hiposmia (ausência ou déficit no sentido do olfato, respectivamente). O HHI tem como principal causa a deficiência do hormônio liberador de gonadotrofina (GnRH), com heterogeneidade genética significativa. Clinicamente é caracterizada por níveis plasmáticos baixos dos hormônios luteinizante (LH) e folículo-estimulante (FSH) associados com baixas concentrações de esteroides sexuais. O estabelecimento de um fenótipo cognitivo claro da SK pode ajudar na compreensão sobre o desenvolvimento das funções cognitivas humanas e a informar para um melhor manejo do paciente, com um acompanhamento mais apropriado. Neste estudo foi realizada uma bateria de avaliação neuropsicológica, caracterizando aspectos referentes aos quocientes intelectuais, atenção, funções executivas, memória, linguagem e habilidades visuoespaciais. Como resultados, o grupo de participantes diagnosticado de SK apresentou desempenho inferior em todas as áreas avaliadas, quando comparado ao grupo controle. Todavia, seu desempenho de maneira geral esteve dentro das faixas média e média-inferior, caracterizando assim um déficit leve das funções neuropsicológicas avaliadas. A memória declarativa de longa duração, no entanto, no grupo clínico esteve bastante prejudicada, com classificação dentro do espectro deficitário. Este resultado, aliado ao desempenho inferior em atividades que exigiam um julgamento de origem temporal e ao comprometimento da aprendizagem de associações condicionais arbitrárias (entre estímulos e respostas), sugere que a maior característica cognitiva presente no grupo clínico está na dificuldade de reter e selecionar informações apropriadas dentre os diversos estímulos e não apenas na recuperação destas informações. / The Kallmann syndrome (KS) is a rare disorder characterized by the presence of hypogonadotropic hypogonadism Idiopathic (IHH) associated with anosmia or hyposmia (absence or deficit in the sense of smell, respectively). The HHI\'s main causes deficiency of gonadotropin-releasing hormone (GnRH), with significant genetic heterogeneity. Clinically, it is characterized by low plasma levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH) associated with low concentrations of sex steroids. The establishment of a clear cognitive phenotype of SK can help in understanding the development of human cognitive functions and to report to a better management of the patient with a more appropriate accompaniment. In this study a battery of neuropsychological evaluation was performed, featuring aspects of intellectual quotients, attention, executive functions, memory, language and visuospatial skills. As a result, the diagnosed group of participants of KS showed lower performance in all areas evaluated, when compared to the control group. However, its performance in general was in the middle and lower-middle ranges, characterizing a slight deficit of assessed neuropsychological functions. The declarative memory of long term, however, in the clinical group was significantly impaired classified within the spectrum deficit. This result, coupled with lower performance in activities requiring a judgment of temporal origin and impairment of learning arbitrary conditional associations (between stimuli and responses), suggests that higher cognitive feature present in the clinical group is the difficulty of retaining and select information appropriate from the various stimuli, not just the recovery of this information.

L’acquisition de la fertilité chez les mammifères est le résultat d’un long processus de développement et de maturation de l’axe gonadotrope. Cette fonction cruciale à la survie des espèces est orchestrée par une poignée de cellules localisées au niveau de l’aire préoptique hypothalamique chez le rongeur, sécrétant la gonadotropin-releasing hormon (GnRH). La GnRH stimule la sécrétion de LH et de FSH par l’adénohypophyse, qui stimulent à leur tour les gonades. Les cellules à GnRH naissent dans l’épithélium voméronasal pendant le développement embryonnaire et migrent le long des axones voméronasaux pour atteindre l’hypothalamus. A la naissance le système est entièrement en place, toutefois il subira une phase de maturation avant d’atteindre la puberté, signant le début de la fertilité. Chez l’homme, un défaut de sécrétion de GnRH peut conduire à un hypogonadisme hypogonadotrope idiopathique (IHH) caractérisé par une subfertilité et une puberté retardée voire absente, ou même à un syndrome de Kallmann. Dans une grande partie des cas ce défaut de sécrétion est lié à un défaut de développement prénatal et à une diminution du nombre de neurones à GnRH dans dans l’hypothalamus. Depuis peu, la grande famille des semaphorines, déjà connues pour leurs effets chimiotactiques dans certains types cellulaires, et en particulier la semaphorine3A (Sema3A) via son récepteur la Neuropilin-1 (Nrp1), a été décrite comme un facteur indispensable au développement du système à GnRH et décrit comme un « gène Kallmann ». Toutefois son rôle spécifique dans les cellules à GnRH reste à élucider. Le premier objectif de ma thèse a donc été de déterminer le rôle de l’expression du récepteur Nrp1 dans les neurones à GnRH. Le suivi de la maturation sexuelle des animaux Gnrh::cre, Nrp1loxp/loxp (qui n’expriment pas la Nrp1 exclusivement dans les neurones à GnRH) a révélé l’apparition d’une puberté précoce et d’un phénotype de surpoids en comparaison aux animaux contrôles, corrélé à une accumulation des cellules à GnRH dans l’aire préoptique. L’étude de l’embryogenèse du système à GnRH chez ces animaux a démontré une augmentation du nombre de cellules à GnRH pendant leur migration. Nos résultats obtenus in vivo et in vitro suggèrent que la signalisation Nrp1 a un impact sur la survie des neurones à GnRH, et qu’elle module la motilité des cellules en migration et influe leur positionnement dans le cerveau. Le deuxième objectif de ma thèse a été d’étudier le rôle de l’exocytose dépendante du calcium et donc de la neurosécrétion dans les neurones à GnRH sur leur développement. Le suivi de la physiologie d’animaux Gnrh::cre; iBot, dont l’exocytose dépendante du calcium est abolit par clivage de protéine VAMP2/synaptobrevin 2 dans le neurone à GnRH, a révélé l’apparition de deux phénotypes distincts selon la pénétrance du transgène : un groupe ayant une puberté normale et un poids comparable aux animaux contrôles, et un groupe ayant une puberté retardée voire inexistante associé à un surpoids. Ces derniers présentent un IHH, une augmentation du tissu adipeux périgonadique et une hyperleptinémie, alors que la distribution des neurones à GnRH dans le cerveau n’est pas altérée. Ces données mettent en évidence le fait que l’activité de neurosécrétion dans les neurones à GnRH ne serait pas nécessaire pour leur développement embryonnaire, mais qu’elle pourrait jouer un rôle dans le maintien de l’homéostasie énergétique.Ces deux études mettent en avant un lien étroit entre axe gonadotrope et métabolisme énergétique chez les mammifères et ont dévoilés de nouveaux mécanismes qui pourraient être impliqués dans la physiopathologie de la reproduction chez l’homme. / Fertility in mammals is the result of a long development and maturation process of the hypothalamic-pituitary-gonadal axis. The reproductive function is orchestrated by a small population of neurons, located in preoptic area of hypothalamus in rodents, and releasing in a pulsatile manner Gonadotropin-releasing hormon (GnRH) in the portal blood vessels, where it is transported to the anterior pituitary gland. GnRH neuropeptide triggers synthesis and release of the gonadotropins LH and FSH, which in turn stimulates development and function of the gonads. GnRH neurons differenciate extracerebraly in the nasal placode and migrate from the vomeronasal organ to the forebrain along olfactory/vomeronasal nerves. At birth, the system is ready, however it will undergo a maturation phase before reaching puberty, signing the beginning of fertility. Deficiency in GnRH release can lead to idiopathic hypogonadotropic hypogonadism (IHH), characterized by a defect in sexual maturation and delayed or no puberty, or even to Kallmann syndrome when the IHH is associated with a deficit in the sens of smell. These phenotypes could be linked to a defect during GnRH neuron migration period and a decrease of GnRH cells located in hypothalamus after birth. Numerous studies have described the influence of different molecules on the migration of GnRH neurons. Recently, the semaphorin family, well known for its chemotactic effects in some cell types, and particularly the semaphorin3A (Sema3A), has been described by our laboratory as an essential factor for the guidance of GnRH neurons during embryogenesis, and characterized as a « Kallmann gene ». However, the role of Sema3A, and its specific receptor Neuropilin-1 (Nrp1) in GnRH neurons remains to be elucidated. The first objective of my thesis was to determine the role of the expression of Nrp1 in GnRH neurons. The analysis of sexual maturation in mice in which Nrp1 expression was selectively knocked out in GnRH neurons revealed a precocious onset of puberty and overweight compared to control littermates, correlated with an accumulation of GnRH neurons in preoptic area. The study of the development of the GnRH system during embryogenesis has shown an increased number of cells during migration. In vivo and in vitro data suggested the involvement of Nrp1 signaling pathway in the survival of GnRH neurons, the control of their motility during migration, and their final positioning in the brain.The second objective of my thesis was to study the role of Calcium-dependent exocytosis, and thus neurosecretion, in GnRH neurons on their development. The monitoring of Gnrh::cre; iBot animals, in which calcium-dependent exocytosis is abolished by cleavage of VAMP2/synaptobrevin2 protein in GnRH neurons, showed the distinction of two different phenotypes. A subpopulation of mice underwent normal puberty onset, with a similar bodyweight than control littermates, and the other one never reached puberty and developed overweight. The later animals exihibited IHH, increase of the volume of perigonadic fat tissue, and hyperleptinemia, with no alteration of GnRH neuron number and distribution. This data established that neurosecretion in GnRH neurons is not a prerequisite for their migration during embryonic development but revealed that it could play an important role in metabolic homeostasis.Together these two studies highlight an intriguing direct connection between GnRH neurons and energy metabolism in mammals as well as new mechanisms that could be implicated in reproductive physiopathology in human.

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

O Hipogonadismo hipogonadotrófico isolado (HHI) congênito é uma síndrome clínica rara causada por defeito na produção ou secreção do hormônio liberador de gonadotrofinas (GnRH) pelo hipotálamo ou por resistência hipofisária à ação do GnRH. O HHI é mais prevalente em homens e cerca de 50% a 60% dos indivíduos afetados apresentam anosmia ou hiposmia associada, caracterizando a síndrome de Kallmann. Diversos genes já foram associados à patogênese do HHI congênito, porém, a maioria dos casos ainda permanece sem diagnóstico molecular definido. Até recentemente, a identificação das causas genéticas dos pacientes com HHI era realizada por sequenciamento de genes candidatos, empregando a técnica de Sanger. No entanto, com o número crescente de genes descritos nos últimos anos, esse processo vem se tornando impraticável. Novas metodologias de sequenciamento (sequenciamento paralelo em larga escala) foram desenvolvidas permitindo a genotipagem simultânea de diversas regiões, com maior velocidade e menor custo relativo. O atual projeto foi desenvolvido com o objetivo de rastrear genes candidatos em pacientes portadores de HHI congênito utilizando-se o sequenciamento paralelo em larga escala, visando ampliar o conhecimento genético do HHI. Realizamos o sequenciamento paralelo em larga escala (SPLE) de 130 pacientes com HHI congênito utilizando um painel contendo 36 genes relacionados ao HHI. Inicialmente, identificamos 104 variantes, potencialmente patogênicas em 77 pacientes (59,2%). Após a filtragem inicial, foi realizada uma análise individualizada de cada variante e com isso foram mantidos 41 (31,5%) pacientes com variantes classificadas como patogênicas ou provavelmente patogênicas. Os genes KAL1, FGFR1, CHD7 e GNRHR foram os mais frequentemente afetados. Esses resultados confirmam a importância dos genes classicamente associados ao HHI congênito. Destaca-se a alta prevalência de variantes no CHD7 (10,8%), gene bastante extenso, levando à dificuldade técnica de sequenciá-lo pelos métodos tradicionais, até então sem estudos nessa coorte. O CHD7 é um gene originalmente associado à complexa síndrome de CHARGE, porém, nos últimos anos vem sendo cada vez mais associados ao HHI congênito. Dentre os resultados, ressaltamos a identificação de uma mutação inédita no gene GNRH1, causa rara de HHI, e a identificação de variantes deletérias no gene IGSF10, recentemente descrito associado ao atraso puberal, mas sem papel claro no fenótipo de HHI, em dois pacientes que tiveram reversibilidade do hipogonadismo. Variantes provavelmente patogênicas em genes com poucas descrições ou até mesmo sem relatos de associação ao fenótipo de HHI (SPRY4, FLRT3, IGSF1, NSMF, SOX10 e OTX2) também foram identificadas nessa coorte, ampliando nosso conhecimento genético do HHI. A oligogenicidade, previamente descrita com prevalência de 2,5% a 7%, em nosso estudo esteve presente em 22% dos pacientes, demonstrando uma ampliação das descrições de oligogenicidade quando comparados aos estudos prévios utilizando somente a técnica de Sanger. A nova técnica de sequenciamento genético (SPLE), utilizada em nosso estudo, foi capaz de ampliar de 22% para 31,5% (41 em 130 pacientes) a porcentagem de pacientes com diagnóstico molecular definido, quando comparado aos dados prévios utilizando a técnica de Sanger, mostrando-se rápida, confiável e eficaz / Congenital isolated hypogonadotropic hypogonadism (IHH) is a rare condition caused by GnRH deficiency, due to defective hypothalamic gonadotropin-releasing hormone (GnRH) production or secretion, or by pituitary resistance to the GnRH action. Congenital IHH is more prevalent in men and about 50% to 60% of affected individuals present with associated anosmia or hyposmia, characterizing Kallmann\'s syndrome. Several genes have already been associated with the pathogenesis of congenital IHH, but most cases still remain without a molecular diagnosis. Until recently, identification of the genetic causes of IHH was performed by sequencing candidate genes using the Sanger technique. However, with the growing number of genes and the genetic complexity of IHH, it has become almost impossible to keep the screening of all candidate genes updated using the traditional techniques. The advent of next-generation sequencing (NGS) has allowed the simultaneous genotyping of several regions, faster and with lower relative cost. The present project was developed with the objective of tracking candidate genes in patients with congenital IHH using large-scale parallel sequencing, in aiming to increase the genetic knowledge of this rare condition. A total of 130 unrelated patients with IHH was studied by targeted NGS, using a panel containing 36 IHH associated genes. Initially, 104 potentially pathogenic variants were identified in 77 patients (59.2%). However, after an individualized analysis of each variant, the number of patients considered to carry pathogenic or probably pathogenic variants dropped to 41 (31.5%). The genes KAL1, FGFR1, CHD7 and GNRHR were the most frequently affected and these results confirm the importance of genes classically associated with IHH. It is noteworthy the high prevalence of variants in CHD7 (10.8%), a rather extensive gene, leading to technical difficulty of sequencing by traditional methods, which had not been studied in this cohort. CHD7 is the causative gene of CHARGE syndrome, however it has been recently identified in a growing number of congenital IHH patients with or without additional features of the syndrome. Among the results, we emphasize a novel mutation in the GNRH1 gene, a rare cause of IHH, and the identification of deleterious variants in the IGSF10 gene, recently associated with pubertal delay but without a clear role in the IHH phenotype, in two patients with reversible hypogonadism. Probably pathogenic variants in genes with few descriptions or even no reports of association with the IHH phenotype (SPRY4, FLRT3, IGSF1, NSMF, SOX10 and OTX2) were also identified in this cohort, increasing the genetic knowledge of IHH. Oligogenicity, previously described with a prevalence of 2.5% to 7%, was observed in 22% of our patients, demonstrating an increase in oligogenicity cases when compared to previous studies using only the Sanger sequencing. In conclusion, targeted NGS was able to increase the percentage of patients with molecular diagnosis from 22% to 31.5% in our cohort when compared to the previous data using the Sanger sequencing, and has been shown to be a fast, reliable and effective tool in the molecular diagnosis of congenital IHH

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.