Les hypogonadismes hypogonadotropes congénitaux (CHH) sont des maladies héréditaires caractérisées par un déficit de sécrétion des gonadotrophines par l’hypophyse, à l’origine d’une infertilité ou d’une absence complète de puberté. On distingue les formes isolées avec olfaction normale (nCHH) et les formes syndromiques associant au déficit gonadotrope d’autres signes, tel qu’un défaut d’olfaction dans le cas du syndrome de Kallmann (SK), la forme plus fréquente de CHH. Les gènes identifiés dans le SK participent au développement embryonnaire et les gènes des nCHH sont impliqués dans la régulation de la sécrétion de la GnRH ou de son action. A ce stade, deux populations de neurones hypothalamiques gonadotropes sont connues, le neurone à GnRH et le neurone KNDy, sécrétant les Kisspeptines et la Neurokinine B. On estimait que l’ensemble des gènes identifiés couvraient moins de 20% des étiologies génétiques. L’objectif de ce doctorat était d’étudier prévalences et mécanismes physiopathologiques des gènes connus et d’identifier de nouvelles étiologies génétiques de CHH. Dans la première partie, nous avons caractérisé la fonctionnalité de tous les variants identifiés sur les gènes KISS1R, TACR3 et TAC3. Cela a permis de préciser les prévalences chez 600 patients, d’identifier un profil neuroendocrinien propre à l’altération de la signalisation Neurokinine B et de démontrer l’implication des Kisspeptines au cours de la vie embryonnaire. Enfin, nous proposons un modèle d’interaction entre le neurone à GnRH et le neurone KNDy. Dans la seconde partie, nous avons identifié deux nouveaux gènes, SEMA3A dans une forme familiale de SK et PNPLA6 dans une forme familiale rare de CHH syndromique. En conclusion, notre connaissance accrue des formes génétiques de CHH, a permis de développer un panel d’exome ciblé dédié au diagnostic par séquençage nouvelle génération permettant l’analyse simultanée de gènes candidats et de gènes connus. / Congenital hypogonadotropic hypogonadism (CHH) is characterized by deficient or absent pubertal development due to deficient or absent secretion of the pituitary gonadotropins. The many known genetic causes are generally classified into distinct nosological groups. One comprises abnormalities that affect the pre-natal development or migration of GnRH neurons, the paradigm of which is Kallmann syndrome. The other encompasses molecular abnormalities that only affect hypothalamic GnRH synthesis, GnRH release or GnRH signaling at pituitary level. At this stage, two populations of hypothalamic neurons implicated in a gonadotrop function are identified, GnRH neurons and KNDy neurons secreting kisspeptins and neurokinin B. All of the identified genes would represent less than 20% of genetic etiologies.The aim of this PhD was to study the prevalence and pathophysiology mechanisms of known genes and to identify new genetic etiologies of CHH.In the first part, we characterized the function of all molecular events identified on KISS1R, TACR3 and TAC3 genes. Prevalences were estimated in 600 patients. A particular neuroendocrine profile was identified in patients presenting an alteration of neurokinin B signaling. Importance of Kisspeptins during embryonic life was validated. According to these data, a model of interaction between GnRH and KNDy neurons was proposed.In the second part, we identified two new CHH genes using various molecular genetics approaches. SEMA3A was identified in a familial form of Kallmann syndrome and PNPLA6 in a rare familial form of CHH.Finally, our increased knowledge of the various genetic forms of CHH allows proposing a new genetic approach based on next generation sequencing to test together all known and several candidate genes.

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.

L’apparition de la puberté et la régulation de la fertilité chez les mammifères sont contrôlées par un réseau neuronal complexe, situé principalement dans l'hypothalamus, et qui converge vers les neurones synthétisant l'hormone de libération des gonadotrophines (GnRH). Ces neurones régulent la sécrétion des gonadotrophines, la croissance et le fonctionnement des gonades. Le développement correct du système à GnRH, incluant des changements rapides dans l'expression et la signalisation de l’hormone GnRH au sein de cette population clairsemée de quelques centaines de neurones, est essentiel pour la maturation sexuelle et le fonctionnement normal de l'axe hypothalamo-hypophyso-gonadique. Lors du développement embryonnaire, ces neurones migrent de la placode olfactive vers leur emplacement définitif, l’hypothalamus, pour y recevoir les connexions afférentes qui permettront une libération pulsatile de la GnRH et la libération subséquente des gonadotrophines (l'hormone de stimulation des follicules (FSH) et l'hormone lutéinisante (LH)). Dès les années 90, l'oxyde nitrique (NO) a été identifié comme molécule clé dans la décharge pré-ovulatoire de GnRH/LH. En effet, de nombreux travaux ont suggéré que des interactions entre les neurones exprimant la forme neuronale de l’enzyme de synthèse du NO (la nNOS) et le système GnRH étaient impliquées dans le contrôle central de la fonction de reproduction à l'âge adulte. De plus, si le NO est reconnu depuis longtemps comme un acteur majeur du contrôle central de l’ovulation à l’âge adulte, la possibilité qu’il soit aussi impliqué dans la maturation sexuelle en régulant l’activité des neurones à GnRH à des stades précoces précédant la puberté n’a pas été explorée auparavant. Cependant, même si nous avons progressé dans la connaissance des interactions entre les neurones à nNOS et des différents acteurs importants de l’axe gonadotrope, l’identité moléculaire de ces neurones reste mal connue. Au cours de cette étude, nous avons recherché 1) l'identité moléculaire des neurones á nNOS dans l'hypothalamus au cours de développement 2) si le NO régule la migration et l’intégration des neurones à GnRH dans l’hypothalamus et 3) si le NO régule la maturation sexuelle. Pendant ma thèse nous avons répertorié pour la première fois les différents neurotransmetteurs et les principaux récepteurs dans les neurones à nNOS au cours du développement post-natal. De plus, les résultats de ma thèse montrent pour la première fois une implication de la signalisation du NO dans la migration des neurones à GnRH vers l'hypothalamus et font échos à l'identification d'une série de mutations de la NOS1 chez des patients atteints du syndrome de Kallmann, une maladie génétique congénitale rare qui associe une carence en GnRH, due à un défaut de migration neuronale, et une anosmie. Enfin, mes travaux montrent que le NO est un nouveau protagoniste dans la maturation post-natale du système à GnRH, la survenue de la puberté et l’acquisition de la capacité à se reproduire. Plus généralement, les résultats de ce travail de thèse permettent d’identifier de nouveaux mécanismes potentiellement responsables de troubles développementaux dans la mise en place des circuits neuronaux contrôlant l’axe gonadotrope chez les mammifères en général et l’homme en particulier. Nous espérons que ces résultats élargiront notre compréhension de la régulation de l'axe reproducteur, offrant ainsi des possibilités nouvelles de stratégies thérapeutiques contre les troubles de la fertilité. / The onset of puberty and the regulation of fertility in mammals are governed by a complex neural network, primarily in the hypothalamus, that converges onto gonadotropin-releasing hormone (GnRH)-producing neurons, the master regulators of gonadotropin secretion and postnatal gonadal growth and function. The proper development of the GnRH system, including timely changes in GnRH expression and signaling by this sparse population of a few hundred neurons, is essential for sexual maturation and the normal functioning of the hypothalamic-pituitary-gonadal axis. As the brain develops during embryogenesis, these neurons should move from the olfactory placode into the correct brain location in adequate numbers, and then establish the afferent connections that will allow the pulsatile release of GnRH peptide, and the subsequent release of the gonadotropins (follicle stimulating hormone, i.e FSH and luteinizing hormone, ie. LH). As early as in the 90’s NO was presented as a key molecule in the preovulatory GnRH/LH surge, and results from different groups, have suggested the interaction of NOS-containing neurons with the GnRH system, and their involvement in the regulation of reproductive capacity. Even though nitric oxide has now been long recognized as a key player in the central hormonal regulation of ovulation during adulthood, no one has considered the possibility that it could act in an earlier stage as the master regulator of GnRH neurons before puberty, hence participating in the actual maturation of the neuroendocrine axis. The relationship of nNOS-expressing neurons with other important molecules of the hypothalamic axis has been well studied, whilst the molecular identity of this neuronal NOS-expressing population is poorly documented. . To this end, we address the hitherto unaddressed questions concerning 1) the molecular identity of nNOS-expressing neurons in the developing hypothalamus, 2) the putative involvement of the NO molecule in the migration of GnRH neurons and the proper establishment of their afferent connections in the hypothalamic region and 3) the plausible determinant role of NO signaling in the maturation of the reproductive system. During this study we identified for the first time the cohort of the principal neurotransmitters and important receptors expressed by these cells in the hypothalamic region during development. Additionally, our results reveal for the first time an involvement of NO signaling in the migration of GnRH neurons in the hypothalamus and are in line with the identification of a series of NOS1 mutations in Kallmann syndrome (KS), a rare congenital genetic condition presenting a unique combination of GnRH deficiency, arising from a faulty migration of the neuronal population, and anosmia. Lastly, our study identifies NO as a novel protagonist during postnatal development, in the regulation of the onset of puberty and the acquisition of reproductive competence. Overall, the results of my Phd thesis identify putative new targets causing alterations of developmental programming under pathophysiological gestational environment in mammals in general, and in humans in particular. Here we thus provide new insights into the mechanisms by which the alteration of GnRH neuronal function leads to hypogonadotropic hypogonadism and infertility. We are hopeful that our results will expand our understanding of how the neuroendocrine axis is regulated and will possibly provide opportunities for therapeutic strategies against debilitating conditions.

Introduction: According to the literature, low serum testosterone levels are associated with diabetes mellitus. Minimal data exist for its prevalence or predictors in South Africa. Erectile dysfunction is a common condition in diabetic patients. The prevalence and predictors in our patient population is unknown. Methods: An observational, cross-sectional study was performed in 150 consecutive male diabetic patients over the age of 50 years in the Diabetic clinic of Steve Biko Academic Hospital. These patients were evaluated for diabetes control and complications, the presence of erectile dysfunction and for hypogonadism symptoms. Morning serum testosterone levels were done. Subjects with low testosterone levels were compared to those with normal levels. Results: The mean age of the patients was 62 years (standard deviation (SD) 7.87), 91.3% had type 2 diabetes, and 84.7% were on insulin. The mean duration of diabetes was 15 years (SD 8.65). The mean body mass index was 30.7 (SD 5.37), the mean waist circumference was 112.4cm (SD 16.42), the median creatinine was 96μmol/L (interquartile range (IQR) 79-133) and the median HbA1C was 7.85% (IQR 6.80-9.30). Ischaemic heart disease was previously diagnosed in 40.7% of patients. Some degree of erectile dysfunction was reported in 95.3% of the patients with 51.3% reporting serious dysfunction. The prevalence of androgen deficiency symptoms was 94.7%. Fifty percent of the men had low total testosterone levels; 40.7% had low modified calculated bioavailable testosterone levels, and in 27.3% both were low. With multivariate logistic regression the significant factors associated with low total testosterone were waist circumference and known cardiovascular disease. For a low modified calculated bioavailable testosterone level significant variables were age, diabetes duration and body mass index and for an outcome defined as both the above the significant factors were diabetes duration, body mass index, and known cardiovascular disease. With multivariate logistic regression the significant factors associated with erectile dysfunction were age, body mass index, peripheral neuropathy score, and diuretic therapy. The prevalence of symptoms of androgen deficiency was very high with 94.7% of all patients reporting a significant amount of symptoms on the Androgen Deficiency in Adult Males (ADAM) questionnaire. If only the total serum testosterone level was evaluated instead of the modified calculated bioavailable testosterone, the sensitivity was 69%, the specificity was 63%, with a poor positive predictive value of only 56%. The negative predictive value was better at 75%. Differences in quality of life scores were only seen for some erectile dysfunction subgroups but not for low testosterone levels. Conclusion: This study confirms the high prevalence of low testosterone levels and of erectile dysfunction in diabetic male patients in a tertiary setting, and argues in favour of universal screening of this population group. Multiple predictors of low testosterone levels and of erectile dysfunction were identified. The ADAM questionnaire was not useful in identifying subjects with a low testosterone level. Total testosterone testing alone performed poorly in comparison with modified calculated bioavailable testosterone and is not the recommended test of choice. Erectile dysfunction negatively affected the quality of life. / Dissertation (MSc)--University of Pretoria, 2014. / gm2015 / Clinical Epidemiology / MSc / Unrestricted

Klinefelter syndrome (KS) (47,XXY) is the most common sex chromosomal anomaly in males with a prevalence of 1 in 650 males. This clinically relevant condition represents 3%-4% of the total patient population in male reproductive medicine practices. Klinefelter syndrome can manifest in different physical, cognitive, and behavioral phenotypes. The classical phenotypic descriptions are gynecomastia, hypotonia, tall stature, and hypogonadism. Currently there is no known treatment plan for adolescents who are diagnosed with this genetic condition. However, it has been hypothesized that early intervention through androgen replacement therapy can emulate a normal progression of puberty and improve the academic, social, and behavioral aspects of these adolescents. Using standardized instruments, in the form of the Children Behavior Checklist (CBCL) and the Youth Self Report (YSR), we captured data on the patient’s competence and behavior, as well as the parents’ views on their child’s competence and behavior. These data were converted into percentile scores, T scores, and categorical data (normal, intermediate, and clinical). Difference of means was used to test for statistically significant differences between the scores of the KS patients and their parents on competence and behavioral aspects. T test for equality of means was run to determine if there was a significant difference between group scores. Pearson correlation tests were done to see if there was an association between demographics of patients and competence/behavioral scores. We believed that there would be no statistically significant difference between how the parents score their child in the CBCL and how the child scores himself in the YSR in terms of demographics. We also believed that there would be no statistically significant difference or correlation between demographics and individual competence/behavioral scores. A total of 39 groups of surveys were collected, together with demographic information on the maternal and paternal ages when the child was born, patient age, patient age when starting testosterone, patient age when starting anastrazole, and patient height and weight. We examined concordance frequencies in specific areas of the competence and behavioral questionnaires. Concordance was when the parent and the child agreed and evaluated the child as normal, intermediate, or clinical. Discordance was when the parents and the child disagreed; for example, the parent evaluated the child as “clinical,” but the child evaluated himself as “normal.” This study, to our knowledge, is the first chart review study that involves a wide age range of males with KS. The study extends previous findings by providing data on how early hormonal intervention can improve the outlook of these patients’ lives. When taking aromatase inhibitors, the KS adolescents showed an overall reduction of aggression and rule-breaking behavior. When taking testosterone at an earlier age, the patient was less likely to have withdrawal depression. We also found the most discordance in total competence scores, suggesting that some parents may have unrealistic academic expectations for their child. Thus, when examining the data, we had to be aware that there were varying views of success, which can shape how parents and child answer their respective surveys. Our findings demonstrate the significance of early detection and treatment of Klinefelter syndrome, a disorder that is severely under-diagnosed throughout the world. The results also show that the desire of parents for their KS child to do well in school may affect their opinions on how their child is actually performing. Some limitations to this study include the decision to receive early hormonal intervention that was made exclusively between parents and their urologist. Furthermore, the socioeconomic status (SES) and educational characteristics of the families were not properly controlled, although the patient population was mostly likely similar because of the location of the clinic. Future Klinefelter syndrome studies will need to focus on identifying the factors that contribute to the variability of behavioral symptoms. In turn, these studies will support the continuing development of evidence-based treatments for adolescents with Klinefelter syndrome.

Mutações inativadoras do receptor de GnRH (GNRHR) são a causa genética mais frequente de hipogonadismo hipogonadotrófico isolado (HHI) normósmico. Os genes envolvidos da patogênese do HHI, incluindo o GNRHR, estão associados a um amplo espectro fenotípico, variando de HHI parcial a completo. O atraso constitucional do crescimento e desenvovimento (ACCD) poderia constituir uma variante fenotípica leve do HHI. Neste estudo avaliamos a frequência de mutações no gene GNRHR em pacientes com HHI normósmico e ACCD, bem como correlacionamos o genótipo/fenótipo nesses pacientes. Além disso, avaliamos o efeito fundador de uma mutação do GNRHR (p.R139H) frequente na população brasileira com HHI normósmico. Para esse estudo, selecionamos 116 pacientes com HHI normósmico e 51 com ACCD. Um grupo de 130 indivíduos com desenvolvimento puberal normal foi utilizado como controle. A região codificadora do gene GNRHR foi amplificada por PCR e sequenciada. Análises in silico e in vitro foram realizadas nas duas novas variantes (p.V134G e p.Y283H). Três marcadores de microssatélites (D4S409, D4S2387, D4S3018) foram amplificados e analisados nos pacientes portadores da mutação p.R139H, familiares e controles. No grupo de HHI normósmico, nove mutações (p.N10K,p.Q11K, p.Q106R, p.R139H, p.C200Y, p.R262Q, p.Y284C, p.Y283H, p.V134G) foram identificadas em onze pacientes (9,5%). Entre as mutações identificadas no GNRHR, duas foram descritas pela primeira vez no estudo atual: p.Y283H e p.V134G, cuja análise in vitro demonstrou inativação completa do receptor. Em geral, uma boa correlação genótipo-fenótipo foi observada. Pacientes portadores de mutações inativadoras apresentavam HHI completo e mutações com perda parcial de função causavam HHI parcial, incluindo dois pacientes que evoluíram com reversão do hipogonadismo após reposição androgênica. Por outro lado, não houve diferença fenotípica entre os casos com e sem mutação do GNRHR. Análise de ancestralidade genética da mutação p.R139H demonstrou que todos os casos brasileiros apresentaram o mesmo haplótipo, sugerindo que a mutação p.R139H possui um ancestral comum na população brasileira. Por outro lado o caso familial proveniente da Polônia apresentou apenas um marcador em comum com as famílias brasileiras e estudos mais abrangentes seriam necessários para determinar a origem da mutação p.R139H em indivíduos não Brasileiros. Na casuística de ACCD apenas a mutação p.Q106R foi identificada no gene GNRHR em heterozigose em um paciente. Em conclusão, o GNRHR foi o gene mais comumente afetado, apresentando uma boa correlação genótipo-fenótipo, e deve ser o primeiro candidato para análise genética em HHI normósmico. Os resultados sugerem que a mutação p.R139H possui um ancestral comum na população brasileira. Mutações no GNRHR parecem não estar envolvidas na patogênese do ACCD / GnRH receptor (GNRHR) inactivating mutations are the most common genetic cause of normosmic IHH. The genes involved in the IHH, including GNRHR, have been associated with a large phenotypic spectrum, varying from partial to complete IHH. Constitutional delay of growth and puberty (CDGP) might represent a mild phenotypic variant of IHH. In this study we investigated novel variants and characterized the frequency and phenotype-genotype correlation of GNRHR mutations in normosmic IHH and CDGP patients. Additionally, we determined de cause of the recurrence of GNRHR p.R139H mutation in patients with normosmic IHH. We studied 116 patients with normosmic IHH and 51 with CDGP. The control group was composed by 130 adults with normal pubertal development. The coding region of GNRHR was amplified and automatically sequenced. The two novel variants identified (p.Y283H, p.V134G) were submitted to in silico and in vitro analysis. Three microsatellite markers (D4S409, D4S2387, D4S3018) were amplified by PCR and analyzed in the patients with the p.R139H mutation. In the CDGP group, the previously described mutation p.Q106R was identified in the heterozygous state in one boy. The p.Q106R mutation has been identified in heterozygous state in individuals with normal pubertal development and does not appear be involved on the CDGP phenotype in this patient. In the normosmic IHH group, nine variants were identified (p.N10K, p.Q11K, p.Q106R, p.R139H, p.C200Y, p.R262Q, p.Y284C, p.Y283H, p.V134G) in eleven patients (9.5%). In vitro analysis of the novel variants p.Y283H and the p.V134G demonstrated that both of them cause complete loss of function of the receptor. The founder effect study revealed that all the p.R139H affected Brazilian patients presented the same haplotype, suggesting that the this mutation has a common ancestor in the Brazilian population. Nevertheless the affected Polish family presented a different haplotype, with only one marker in common with the Brazilian families and further studies would be necessary to determine the origin of the p.R139H mutation in the European population. In conclusion this study demonstrated that GNRHR was the most commonly affected gene in normosmic IHH, with a good genotype-phenotype correlation, and should be the first candidate gene for genetic screening in this condition. The results of the founder effect study suggested that the p.R139H mutation has a common ancestor in the Brazilian population. Finally, mutations in the GNRHR do not appear to be involved in the pathogenesis of CDGP

Σκοπός της μελέτης ήταν η διερεύνηση ύπαρξης μεταλλάξεων στα γονίδια KAL, της GnRH, του υποκινητή της GnRH, του υποδοχέα της GnRH, και του υποκινητή του υποδοχέα της GnRH, σε ασθενείς με ανεπάρκεια GnRH, με στόχο την εξαγωγή συμπερασμάτων σχετικά με τη συχνότητα των διαφόρων μορφών μετάδοσης της νόσου στον ελληνικό χώρο, καθώς και η συσχέτιση μεταξύ του γονότυπου των ασθενών και ειδικών κλινικών φαινοτύπων. Μελετήθηκαν συνολικά τριάντα οκτώ (38) ασθενείς με ανεπάρκεια GnRH, δώδεκα (12) ασθενείς με σύνδρομο Kallmann και είκοσι έξι (26) ασθενείς (13 άνδρες και 13 γυναίκες) με ιδιοπαθή υπογοναδοτροφικόΤο σύνδρομο ανεπάρκειας της GnRH περιλαμβάνει ετερογενή, αλλά συναφή προς το κλινικό φαινότυπο πληθυσμό ασθενών οι οποίοι παρουσιάζουν πλήρη ή μερική απώλεια της ικανότητας του οργανισμού τους να προάγει την κατά ώσεις έκκριση της GnRH. Η ανεπάρκεια αυτή οδηγεί στην πλήρη ή μερική αναστολή της σεξουαλικής ωρίμανσης και σε στειρότητα του ασθενούς. Η παρουσία συνοδού ανοσμίας αναφέρεται ως σύνδρομο Kallmann, ενώ η απουσία άλλων συνοδών ανωμαλιών ως ιδιοπαθής υπογοναδοτροφικός υπογοναδισμός (ΙΥΥ). Σκοπός της μελέτης ήταν η διερεύνηση ύπαρξης μεταλλάξεων στα γονίδια KAL, της GnRH, του υποκινητή της GnRH, του υποδοχέα της GnRH, και του υποκινητή του υποδοχέα της GnRH, σε ασθενείς με ανεπάρκεια GnRH, με στόχο την εξαγωγή συμπερασμάτων σχετικά με τη συχνότητα των διαφόρων μορφών μετάδοσης της νόσου στον ελληνικό χώρο, καθώς και η συσχέτιση μεταξύ του γονότυπου των ασθενών και ειδικών κλινικών φαινοτύπων. Μελετήθηκαν συνολικά τριάντα οκτώ (38) ασθενείς με ανεπάρκεια GnRH, δώδεκα (12) ασθενείς με σύνδρομο Kallmann και είκοσι έξι (26) ασθενείς (13 άνδρες και 13 γυναίκες) με ιδιοπαθή υπογοναδοτροφικό υπογοναδισμό (ΙΥΥ). Η μεθοδολογία της εργαστηριακής έρευνας περιέλαβε απομόνωση DNA γονιδιώματος από τους ασθενείς εκλεκτικό πολλαπλασιασμό της κωδικοποιησης με την μέθοδο PCR και τέλος προσδιορισμό της αλληλουχίας του DNA στα προϊόντα της PCR. Στους ασθενείς με σύνδρομο Kallmann δεν ανευρέθη αλλαγή βάσεων του γονιδίου KAL. Επισης, στους ασθενείς με ιδιοπαθή υπογοναδοτροφικό υπογοναδισμό δεν εντοπίστηκαν μεταλλάξεις στους υποκινητές των γονιδίων της GnRH και του υποδοχέα της. Σε πέντε ασθενείς με ιδιοπαθή υπογοναδοτροφικό υπογοναδισμό που αφορούσαν σποραδικές περιπτώσεις, εντοπίστηκε η μετάλλαξη (gc στο κωδικόνιο 16 Trp16Ser ) στο γονίδιο της GnRH η οποία αποτελεί φυσικό πολυμορφισμό. Στο γονίδιο του υποδοχέα της GnRH εντοπίστηκαν δύο μεταλλάξεις. Η μετάλλαξη (ct στο κωδικόνιο 146), ανιχνεύτηκε σε δύο ασθενείς με οικογενή κληρονομικότητα. Η μετάλλαξη αυτή προκαλεί αλλαγή του αμινοξέως στη θέση 146 της πρωτείνης από προλίνη σε σερίνη την οποία φέρουν και οι δύο ασθενείς σε ετεροζυγωτία. Στους δύο ασθενείς με ΙΥΥ και τη μετάλλαξη Pro146Ser, καθώς και σε ένα άνδρα με ΙΥΥ και φυσιολογική αλληλουχία του υποδοχέα της GnRH παρατηρήθηκε αντίσταση στη δράση της GnRH. Ασθενείς με ΙΥΥ και αντίσταση στη δράση της GnRH αποτελούν φυσικά πρότυπα για τη μελέτη και τον εντοπισμό των πολλαπλών γονοτυπικών συνδυασμών οι οποίοι έχουν ως κατάληξη την εμφάνιση του συγκεκριμένου φαινοτύπου. Ο μη εντοπισμός νοσογόνων μεταλλάξεων ομοζυγωτίας ή διπλής ετεροζυγωτίας στους συγκεκριμένους ασθενείς συνηγορεί στην ύπαρξη διαταραχών στην έκφραση γονιδίων τα οποία επηρεάζουν ή την ενδοκυττάρια μετάδοση του μηνύματος ή την ίδια την έκφραση του υποδοχέα της GnRH. Αν και το γονίδιο KAL έχει ενοχοποιηθεί για την ανάπτυξη ψυχοπαθολογικών εκδηλώσεων, με κύριο εκπρόσωπο τη σχιζοφρένεια, οι εκδηλώσεις από τη ψυχική σφαίρα ασθενών με σύνδρομο Kallmann δεν έχουν μελετηθεί. Επιπλέον σκοπός της παρούσης μελέτης ήταν να περιγράψει αυτές τις διαταραχές και να τις συσχετίσει με το γονότυπο των ασθενών. Ένας εκ των ασθενών με σύνδρομο Kallmann παρουσίαζε και σχιζοφρένεια χωρίς όμως να αναδειχθούν μεταλλάξεις στο γονιδίωμά του. Ενώ παράλληλα οι ασθενείς με ανεπάρκεια GnRH δεν διαφέρουν σημαντικά στις κλίμακες ψυχοπαθολογίας ούτε από τις μέσες τιμές φυσιολογικού πληθυσμού, ούτε από τις τιμές που έδωσε η ομάδα ελέγχου με χρόνιες σωματικές παθήσεις, δίνουν σημαντικά χαμηλότερες τιμές σε όλες τις κλίμακες ψυχοπαθολογίας από ότι οι ψυχιατρικά ασθενείς, εκτός από την υποκλίμακα του «θυμού». / The syndrome of GnRH insufficiency is due to a functional deficit of GnRH production or secretion in the hypothalamus resulting in the loss of pulsatile secretion of GnRH. This deficiency leads to a complete or partial arrest of sexual maturation and infertility. Patients with no further anomalies are referred as having Idiopathic Hypogonadotropic Hypogonadism (IHH) and when accompanied with anosmia, it is called Kallmann syndrome. The aim of this study was to identify mutations in the KAL gene, the GnRH gene, the GnRH receptor gene and their promoters in patients with GnRH insufficiency, the prevalence in the Greek population and the relevance between the genotype and individual phenotype of these patients. The study included thirty eight (38) patients with GnRH insufficiency, twelve patients (12) with Kallmann syndrome and twenty six patients with IHH (13 male and 13 female). Detection was carried out by isolation of genomic DNA from whole blood, which was used as a template for PCR amplification and finally, cycle sequencing analysis of all exons spanning the entire coding regions of the genes. No mutations were found in the KAL gene, whereas in patients with IHH, no mutations were identified in transcription factor binding sites of the promoters of the GnRH and GnRH receptor gene. In the GnRH gene of five (5) patients with IHH a natural polymorphism was identified in codon 16 (gc Trp16Ser). In the GnRH receptor gene a novel mutation was found in codon 146, resulting in substitution of proline with serine identified in two sisters harboring this mutation in hetrozygosity. The two sisters and a male patient with IHH with normal gene sequencing were found to be resistant to GnRH action. Resistance to GnRH is particularly rare among IHH patients. One might hypothesize, that these patients ought to have inactivating mutations in their receptor gene. However such a defect was not found, therefore making these patients an ideal clinical phenotype of an aberration in signal transduction pathway or in transcriptional factors which regulate the expression of the GnRH receptor. A common pathogenesis for KS and schizophrenia had been proposed, based on shared pathologies of these two disorders. The gene for the X-linked form of KS (known as KAL) has been implicated in the genetic pathogenesis of schizophrenias, although no such clinical associations have ever been reported. An additional aim of this study was to identify any pshychopathologies in these patients. One of our patients with KS also developed schizophrenia but no mutations were identified in all 14 exons of the KAL gene.

Mutações inativadoras do receptor de GnRH (GNRHR) são a causa genética mais frequente de hipogonadismo hipogonadotrófico isolado (HHI) normósmico. Os genes envolvidos da patogênese do HHI, incluindo o GNRHR, estão associados a um amplo espectro fenotípico, variando de HHI parcial a completo. O atraso constitucional do crescimento e desenvovimento (ACCD) poderia constituir uma variante fenotípica leve do HHI. Neste estudo avaliamos a frequência de mutações no gene GNRHR em pacientes com HHI normósmico e ACCD, bem como correlacionamos o genótipo/fenótipo nesses pacientes. Além disso, avaliamos o efeito fundador de uma mutação do GNRHR (p.R139H) frequente na população brasileira com HHI normósmico. Para esse estudo, selecionamos 116 pacientes com HHI normósmico e 51 com ACCD. Um grupo de 130 indivíduos com desenvolvimento puberal normal foi utilizado como controle. A região codificadora do gene GNRHR foi amplificada por PCR e sequenciada. Análises in silico e in vitro foram realizadas nas duas novas variantes (p.V134G e p.Y283H). Três marcadores de microssatélites (D4S409, D4S2387, D4S3018) foram amplificados e analisados nos pacientes portadores da mutação p.R139H, familiares e controles. No grupo de HHI normósmico, nove mutações (p.N10K,p.Q11K, p.Q106R, p.R139H, p.C200Y, p.R262Q, p.Y284C, p.Y283H, p.V134G) foram identificadas em onze pacientes (9,5%). Entre as mutações identificadas no GNRHR, duas foram descritas pela primeira vez no estudo atual: p.Y283H e p.V134G, cuja análise in vitro demonstrou inativação completa do receptor. Em geral, uma boa correlação genótipo-fenótipo foi observada. Pacientes portadores de mutações inativadoras apresentavam HHI completo e mutações com perda parcial de função causavam HHI parcial, incluindo dois pacientes que evoluíram com reversão do hipogonadismo após reposição androgênica. Por outro lado, não houve diferença fenotípica entre os casos com e sem mutação do GNRHR. Análise de ancestralidade genética da mutação p.R139H demonstrou que todos os casos brasileiros apresentaram o mesmo haplótipo, sugerindo que a mutação p.R139H possui um ancestral comum na população brasileira. Por outro lado o caso familial proveniente da Polônia apresentou apenas um marcador em comum com as famílias brasileiras e estudos mais abrangentes seriam necessários para determinar a origem da mutação p.R139H em indivíduos não Brasileiros. Na casuística de ACCD apenas a mutação p.Q106R foi identificada no gene GNRHR em heterozigose em um paciente. Em conclusão, o GNRHR foi o gene mais comumente afetado, apresentando uma boa correlação genótipo-fenótipo, e deve ser o primeiro candidato para análise genética em HHI normósmico. Os resultados sugerem que a mutação p.R139H possui um ancestral comum na população brasileira. Mutações no GNRHR parecem não estar envolvidas na patogênese do ACCD / GnRH receptor (GNRHR) inactivating mutations are the most common genetic cause of normosmic IHH. The genes involved in the IHH, including GNRHR, have been associated with a large phenotypic spectrum, varying from partial to complete IHH. Constitutional delay of growth and puberty (CDGP) might represent a mild phenotypic variant of IHH. In this study we investigated novel variants and characterized the frequency and phenotype-genotype correlation of GNRHR mutations in normosmic IHH and CDGP patients. Additionally, we determined de cause of the recurrence of GNRHR p.R139H mutation in patients with normosmic IHH. We studied 116 patients with normosmic IHH and 51 with CDGP. The control group was composed by 130 adults with normal pubertal development. The coding region of GNRHR was amplified and automatically sequenced. The two novel variants identified (p.Y283H, p.V134G) were submitted to in silico and in vitro analysis. Three microsatellite markers (D4S409, D4S2387, D4S3018) were amplified by PCR and analyzed in the patients with the p.R139H mutation. In the CDGP group, the previously described mutation p.Q106R was identified in the heterozygous state in one boy. The p.Q106R mutation has been identified in heterozygous state in individuals with normal pubertal development and does not appear be involved on the CDGP phenotype in this patient. In the normosmic IHH group, nine variants were identified (p.N10K, p.Q11K, p.Q106R, p.R139H, p.C200Y, p.R262Q, p.Y284C, p.Y283H, p.V134G) in eleven patients (9.5%). In vitro analysis of the novel variants p.Y283H and the p.V134G demonstrated that both of them cause complete loss of function of the receptor. The founder effect study revealed that all the p.R139H affected Brazilian patients presented the same haplotype, suggesting that the this mutation has a common ancestor in the Brazilian population. Nevertheless the affected Polish family presented a different haplotype, with only one marker in common with the Brazilian families and further studies would be necessary to determine the origin of the p.R139H mutation in the European population. In conclusion this study demonstrated that GNRHR was the most commonly affected gene in normosmic IHH, with a good genotype-phenotype correlation, and should be the first candidate gene for genetic screening in this condition. The results of the founder effect study suggested that the p.R139H mutation has a common ancestor in the Brazilian population. Finally, mutations in the GNRHR do not appear to be involved in the pathogenesis of CDGP

Orientador: Vania dos Santos Nunes Nogueira / Resumo: Introdução: o uso de testosterona em homens obesos tem sido controverso em termos de perda de peso e controle de doenças relacionadas à obesidade. Objetivo: avaliar a efetividade da reposição de testosterona para perda de peso e prevenção de complicações cardiovasculares em homens obesos com baixos níveis séricos de testosterona. Métodos: foi realizada uma revisão sistemática de acordo com a Metodologia Cochrane de estudos randomizados que compararam em homens obesos com baixos níveis de testosterona a reposição de testosterona versus a não reposição. Dois revisores (ASM e LARB) independentemente fizeram a seleção dos estudos, avaliaram o risco de viés e extraíram os dados dos estudos incluídos. Os desfechos primários analisados foram: melhora dos parâmetros antropométricos (peso, índice de massa corpórea, circunferência de cintura e composição corporal), segurança, qualidade de vida, controle de complicações relacionadas a obesidade e melhora dos sintomas de hipogonadismo. As bases de dados eletrônicas para pesquisa foram Embase, Medline, LILACS e CENTRAL. Desfechos semelhantes em pelo menos dois estudos foram plotados na meta-análise usando o software Review Manager 5.3. A qualidade da evidência da estimativa de efeito da intervenção foi gerada de acordo com a metodologia GRADE (Grading of Recommendations Assessment, Development and Evaluation). Resultados: as estratégias de busca elaboradas resultaram em 2535 artigos após a remoção das duplicatas pelo software Endnote. For... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Introduction: The use of testosterone replacement therapy in obese men with low testosterone levels has been controversial in relation to weight loss and control of obesity related-diseases. Objective: To analyze the effectiveness of testosterone therapy for weight loss and prevention of cardiovascular complications in obese men with low serum testosterone levels. Methods: A systematic review was conducted according to the Cochrane Methodology of randomized trials comparing testosterone replacement versus non-replacement in obese men with low testosterone levels. Two reviewers (ASM and LARB) independently selected the studies, assessed the risk of bias, and extracted data from the included studies. The main outcomes analyzed were: improvement of anthropometric parameters (weight, body mass index, waist circumference and body composition), safety, quality of life, control of complications related to obesity and improvement of symptoms of hypogonadism. The electronic databases for search were Embase, Medline, LILACS and CENTRAL. Similar outcomes in at least two trials were plotted in the meta-analysis using Review Manager 5.3 software. The quality of the evidence of the intervention effect estimate was generated according to GRADE approach (Grading of Recommendations Assessment, Development and Evaluation). Results: the search strategies elaborated resulted in 2535 articles after the duplicates were removed by Endnote software. A total of 38 articles were selected for reading, ... (Complete abstract click electronic access below) / Mestre

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.