L’infertilité représente un problème majeur de santé publique en concernant 10 à 15% des couples en âge de procréer. Un facteur masculin est responsable de l’infertilité du couple dans près de la moitié des cas. Pour environ 30% d'entre eux, l'étiologie reste inexpliquée. Le premier axe du travail a concerné l’étude moléculaire d’une cohorte de patients infertiles (azoospermie non-obstructive/cryptozoospermie ou désordre du développement sexuel ou DSD) pour lesquels les analyses du caryotype standard et/ou des microdélétions des régions AZF par PCR n’ont pas permis d’expliquer le phénotype. L'impact des variations de nombre de copies de l'ADN (CNV) détectées par l'hybridation génomique comparative sur puce à ADN est peu documenté. Un design personnalisé de puce à ADN de format 400K, pangénomique et enrichi sur un large panel de 445 gènes liés à l'infertilité et à un DSD a été développé. Cette puce a permis l’identification de 171 CNV d’intérêt. Ces résultats soulignent l’intérêt de ce design comme outil diagnostic dans le cadre du bilan de l’infertilité masculine. Le second axe du travail a été de modéliser l’infertilité masculine in vitro dans un contexte d’anomalie génétique. Des cellules souches pluripotentes induites humaines (hiPS) ont été générées à partir d’érythroblastes de deux patients infertiles porteurs d’un remaniement chromosomique complexe ou d’un caryotype 46,XX-SRY négatif avec mutation du gène de l’AMH. Dans un deuxième temps, la fonctionnalité des lignées de cellules hiPS générées a été testée par différenciation in vitro en cellules germinales primordiales (CGP). Elles expriment les marqueurs clés du stade CGP dont SOX17, le déterminant germinal le plus précoce des CGP. Les perspectives de ce travail seront de poursuivre la différenciation germinale vers des stades plus matures et ainsi de pouvoir étudier le processus méiotique dans un contexte d’anomalie génétique. / Infertility represents a major public health problem and concerns 10 to 15% of couples in the general population. A male factor is responsible for the infertility of the couple in about half of all cases. In approximately 30% of them, the etiology remains unexplained.The first working axis concerned the molecular study of a cohort of infertile patients (nonobstructiveazoospermia/ cryptozoospermia and disorder of the sex development or DSD) for whom analyses of standard karyotype and/or microdeletions of AZF regions were not able to explain the phenotype. The impact of copy number variations of DNA (CNVs) detected by comparative genomic hybridization (CGH-array) is poorly documented. A custom design 400K micoarray, genome-wide and enriched on a wide panel of 445 genes linked with infertility and DSD has been achieved. This array allowed the identification of 171 CNVs of interest.These results underline the potential of this design for diagnosis of male infertility. The second objective of this work was the in vitro modelisation of male infertility in a context of genetic abnormality. For that purpose, human induced pluripotent stem cells (hiPSCs) were generated from erythroblasts by means of not integrative Sendaï virus, in two patients carrying genetic abnormalities (complex chromosomal rearrangement and 46,XX-SRY negative karyotype associated with AMH gene mutation). Secondly, functionality of hiPSCs generated was tested by germ cells in vitro differentiation. Primordial germ cell (PGC) stage was successfully obtained. Cells expressed key PGC markers such as SOX17. The perspectives of this work will be to continuethe germinal differentiation towards more mature stages and so to be able studying the meiotic process in a context of genetic abnormality.

The DSM-IV-TR diagnosis Gender Identity Disorder Not Otherwise Specified (GID NOS) is used to describe individuals who have gender issues but do not meet the current criteria for GID. As part of a consensus process conducted by the World Professional Association for Transgender Health, the authors make the following recommendations for DSM 5: removal from the chapter on sexual disorders, more specific diagnostic criteria, retention of clinical significance criteria, and removal of exclusionary criteria of Intersex/Disorders of Sex Development. Changes to the existing clinical examples were also recommended, suggesting additional clinical examples that encompass a broader range of gender-variance and more commonly found transgender presentations. The diagnosis must reflect the severity of the clinical issues that represent legitimate identity experiences and possible need for gender-confirming treatments.

Against a backdrop of feminist and social scientific research on sex, female embodiment, and normality this thesis aims to discern how young women, who in adolescence have learned that their bodies are developing in ways considered ‘atypical’ for the female sex, make sense of their bodies and their situation. In focus are the ways in which the women make sense of and negotiate female embodiment; how they, particularly in stories about their interactions with others, position their embodied selves; and how norms and beliefs about sexed embodiment, heterosexual practice, and in/fertility are strengthened and challenged in the interviewees’ sense-making. The data comprise 23 in-depth interviews with women who in adolescence have learned that they do not have a uterus and a vagina, or who have learned that they do not have two X chromosomes and have no, or non-functioning, ovaries. Through narrative and thematic analysis the thesis shows how the women’s sense-making can be obstructed by norms about female embodiment, heterosexual practice, and in/fertility, expressed through medical terminology and practice and in interaction with family, friends, and peers, as described by the interviewees. Concomitantly, as the thesis demonstrates, medical terminology can be experienced and function as a resource in the women’s sense-making. Diagnostic categories enable them to put the specificities of sex development into words and raise awareness about bodily variation. Furthermore, in their stories about others’ reactions to their bodies and about their experience and management of certain medical treatments, the women question norms about female embodiment, heterosexual practice, and in/fertility that were previously taken for granted. The complexity of the women’s sense-making is demonstrated through the ways in which the interviewees, on the one hand, align with norms about female embodiment, heterosexual practice, and in/fertility, and in which they, on the other hand, succeed in challenging the same. In this ‘juggling’ of reinforcement and resistance, the thesis argues, the women are found to expand rather than dismiss beliefs about female embodiment.  Thus, the thesis contributes with deepened knowledge about what it can be like to live with these specific conditions and with problematizations of how norms about female embodiment can be enacted and questioned. / Mot bakgrund av feministisk och samhällsvetenskaplig forskning kring kön, kvinnlig kroppslighet och normalitet syftar avhandlingen till att undersöka hur unga kvinnor, som i tonåren fått reda på att deras kropp utvecklas på ett sätt som anses ”otypiskt” för det kvinnliga könet söker förstå och skapa mening kring sin kropp och situation. Framförallt undersöks dessa kvinnors meningsskapande, hur de i sina berättelser positionerar sig i relation till andra, och hur normer och föreställningar om kvinnlig kroppslighet, heterosexuell praktik och in/fertilitet förstås, förhandlas, stärks och ifrågasätts i berättelserna. Materialet som undersöks utgörs av 23 djupintervjuer med kvinnor som i tonåren fått reda på att de antingen inte har någon livmoder och vagina eller att de inte har två X kromosomer och inga eller  icke-fungerade äggstockar. Genom narrativa och tematiska analyser visar avhandlingen hur kvinnornas meningskapande formas av normer kring kvinnlig kroppslighet, heterosexuell praktik och in/fertilitet, då de uttrycks i kvinnornas berättelser om sin situation i möten med andra och i relation till medicinsk praktik. Samtidigt, visar avhandlingen, kan medicinsk terminologi, specifikt diagnoser, och praktik utgöra resurser i kvinnornas meningsskapande som möjliggör för dem att sätta ord på och sprida kunskap om kroppslig variation. I kvinnornas berättelser om andras reaktioner på deras kroppar och om deras erfarenhet och hantering av specifika medicinska behandlingar utmanas vidare normer som kvinnorna tidigare har tagit för givet. Genom analysen framträder således komplexiteten i kvinnornas meningskapande då de å ena sidan anammar förgivettagna normer om kvinnlig kroppslighet, heterosexuell praktik och infertilitet och å andra sidan utmanar de samma. I detta ”jonglerande” av anpassning till normer och motstånd mot desammasyns kvinnorna expandera snarare än avfärda föreställningar om kvinnlig kroppslighet. Avhandlingen fördjupar därmed kunskapen om hur det kan vara att leva med dess specifika tillstånd och till att problematisera hur normer om kvinnlig kroppslighet kan ta sig uttryck och ifrågasättas.

O objetivo deste estudo foi avaliar a influência da exposição a níveis normais ou reduzidos de testosterona durante a vida intrauterina no desenvolvimento psicológico, social e sexual dos pacientes com DDS 46, XY. Pacientes e métodos: Trata-se de um estudo retrospectivo. Os 53 participantes são pacientes portadores de DSD 46,XY devido a defeitos de produção de testosterona ou deficiência da 5alfa-RD2, todos eles com genitália ambígua que resultou na atribuição do sexo feminino ao nascimento. Os pacientes foram divididos em dois grupos: Grupo 1 (G1) - pacientes com DDS 46, XY, devido a defeito na produção de testosterona, constituído por 29 pacientes, 8 deles com deficiência de 17beta-HSD3, 7 com hipoplasia das células de Leydig, 7 com disgenesia gonadal parcial, 6 com deficiência 17alfa-hidroxilase e 1 com deficiência 3beta-HSD2; Grupo 2 (G2) - constituído por 24 pacientes com deficiência de 5alfa-RD2. Foi utilizado um questionário com 32 perguntas abrangendo aspectos do desenvolvimento psicológico, social e sexual destes pacientes. Resultados: Foi encontrada uma diferença significativa nos seguintes aspectos do desenvolvimento psicológico, social e sexual dos participantes do estudo: maior incidência de masturbação, fantasias eróticas e desejo de ter filhos em pacientes com deficiência da 5alfa-RD2 com sexo social masculino. Nas pacientes com sexo social feminino o desejo de ter filhos foi maior naquelas com DDS 46, XY por defeitos na produção de testosterona do que naquelas com deficiência da 5alfa-RD2 (p < 0,05), enquanto que o desejo de ter filhos foi maior nos homens com deficiência 5alfa-RD2 (p > 0,05). O número de indivíduos casados foi significativamente maior no grupo dos pacientes com DDS 46, XY por defeitos na produção de testosterona do que no grupo dos pacientes com DDS 46, XY por deficiência da 5alfa-RD2 (p = 0,003). Em conclusão, nossos resultados indicam uma possível influência da exposição aos andrógenos durante a vida pré-natal no desenvolvimento psicológico e social, bem como em aspectos da vida sexual dos pacientes adultos com DDS 46, XY / The aim of this study was to evaluate the influence of exposure to normal or reduced levels of testosterone during intra-uterine life in psychological, social, and sexual development of patients with DSD 46, XY. Patients and methods: This is a retrospective study. The 53 participants were patients with DSD 46, XY due to defects in production of testosterone or deficiency of 5alfa-RD2, all of them with ambiguous genitalia and female sex assignment at birth. These patients were divided into two groups: Group 1 (G1) - patients with DSD 46, XY, due to a defect in the production of testosterone, consisting of 29 people, 8 with deficiency of 17beta-HSD3, 7 with Leydig cell hypoplasia, 7 with partial gonadal dysgenesis, 6 with 17alfa-hydroxylase deficiency ,1 with 3beta-HSD2 deficiency, Group 2 (G2) - consisting of 24 patients with deficiency of 5alfa-RD2. We used a questionnaire with 32 questions covering aspects of psychological, social and sexual development of these patients. Results: A significant difference was found in the following aspects of psychological, social and sexual development of these patients: higher incidence of masturbation, erotic fantasies and desire for children in patients with deficiency of 5alfa-RD2 with male social sex. In patients with female social sex, the desire to have children was higher in those with DSD 46, XY by defects in the production of testosterone than in those with deficiency of 5alfa-RD2 (p < 0.05), while the desire to have children in men was higher in those with 5alfa-RD2 (p > 0.05). The number of married individuals was significantly higher in the group of patients with DSD 46, XY by defects in the production of testosterone than in the group of patients with DSD 46, XY by deficiency of 5alfa-RD2 (p = 0.003). In conclusion, our results indicate a possible influence of exposure to androgens during prenatal life in psychological and social development, as well as in aspects of sexual life of adult patients with DSD 46, XY

O objetivo deste estudo foi avaliar a influência da exposição a níveis normais ou reduzidos de testosterona durante a vida intrauterina no desenvolvimento psicológico, social e sexual dos pacientes com DDS 46, XY. Pacientes e métodos: Trata-se de um estudo retrospectivo. Os 53 participantes são pacientes portadores de DSD 46,XY devido a defeitos de produção de testosterona ou deficiência da 5alfa-RD2, todos eles com genitália ambígua que resultou na atribuição do sexo feminino ao nascimento. Os pacientes foram divididos em dois grupos: Grupo 1 (G1) - pacientes com DDS 46, XY, devido a defeito na produção de testosterona, constituído por 29 pacientes, 8 deles com deficiência de 17beta-HSD3, 7 com hipoplasia das células de Leydig, 7 com disgenesia gonadal parcial, 6 com deficiência 17alfa-hidroxilase e 1 com deficiência 3beta-HSD2; Grupo 2 (G2) - constituído por 24 pacientes com deficiência de 5alfa-RD2. Foi utilizado um questionário com 32 perguntas abrangendo aspectos do desenvolvimento psicológico, social e sexual destes pacientes. Resultados: Foi encontrada uma diferença significativa nos seguintes aspectos do desenvolvimento psicológico, social e sexual dos participantes do estudo: maior incidência de masturbação, fantasias eróticas e desejo de ter filhos em pacientes com deficiência da 5alfa-RD2 com sexo social masculino. Nas pacientes com sexo social feminino o desejo de ter filhos foi maior naquelas com DDS 46, XY por defeitos na produção de testosterona do que naquelas com deficiência da 5alfa-RD2 (p < 0,05), enquanto que o desejo de ter filhos foi maior nos homens com deficiência 5alfa-RD2 (p > 0,05). O número de indivíduos casados foi significativamente maior no grupo dos pacientes com DDS 46, XY por defeitos na produção de testosterona do que no grupo dos pacientes com DDS 46, XY por deficiência da 5alfa-RD2 (p = 0,003). Em conclusão, nossos resultados indicam uma possível influência da exposição aos andrógenos durante a vida pré-natal no desenvolvimento psicológico e social, bem como em aspectos da vida sexual dos pacientes adultos com DDS 46, XY / The aim of this study was to evaluate the influence of exposure to normal or reduced levels of testosterone during intra-uterine life in psychological, social, and sexual development of patients with DSD 46, XY. Patients and methods: This is a retrospective study. The 53 participants were patients with DSD 46, XY due to defects in production of testosterone or deficiency of 5alfa-RD2, all of them with ambiguous genitalia and female sex assignment at birth. These patients were divided into two groups: Group 1 (G1) - patients with DSD 46, XY, due to a defect in the production of testosterone, consisting of 29 people, 8 with deficiency of 17beta-HSD3, 7 with Leydig cell hypoplasia, 7 with partial gonadal dysgenesis, 6 with 17alfa-hydroxylase deficiency ,1 with 3beta-HSD2 deficiency, Group 2 (G2) - consisting of 24 patients with deficiency of 5alfa-RD2. We used a questionnaire with 32 questions covering aspects of psychological, social and sexual development of these patients. Results: A significant difference was found in the following aspects of psychological, social and sexual development of these patients: higher incidence of masturbation, erotic fantasies and desire for children in patients with deficiency of 5alfa-RD2 with male social sex. In patients with female social sex, the desire to have children was higher in those with DSD 46, XY by defects in the production of testosterone than in those with deficiency of 5alfa-RD2 (p < 0.05), while the desire to have children in men was higher in those with 5alfa-RD2 (p > 0.05). The number of married individuals was significantly higher in the group of patients with DSD 46, XY by defects in the production of testosterone than in the group of patients with DSD 46, XY by deficiency of 5alfa-RD2 (p = 0.003). In conclusion, our results indicate a possible influence of exposure to androgens during prenatal life in psychological and social development, as well as in aspects of sexual life of adult patients with DSD 46, XY

Le développement sexuel est un processus complexe qui dépend de nombreux gènes, une mutation pouvant entraîner un développement sexuel anormal. Par ailleurs, des anomalies chromosomiques peuvent avoir des répercussions importantes sur la détermination gonadique, surtout lorsqu'il s'agit du chromosome Y puisqu'il porte le gène clé du développement sexuel masculin. Premièrement, nous avons identifié par cytogénétique moléculaire le point de cassure chez 5 patients avec une translocation X;Y et 10 patients avec un chromosome Y isodicentrique. Nous avons ainsi démontré que certaines régions sont plus à risque d'être remaniées, notamment lorsqu'elles contiennent des palindromes ou d'autres séquences répétées. Nous avons également établi une relation entre la distance séparant le centromère et le point de cassure et l'instabilité des chromosomes Y isodicentriques lors des divisions cellulaires. Deuxièmement, nous avons étudié en cytogénétique les gonades de 22 patients avec un chromosome Y normal ou remanié et présentant un développement sexuel anormal. Nous avons mis en évidence la perte du chromosome Y remanié dans une majorité de cellules gonadiques des 10 patients étudiés, expliquant leur phénotype sexuel anormal. Cependant, chez 11 des 12 patients avec un chromosome Y normal, aucun mosaïcisme expliquant clairement leur détermination gonadique anormale n'a été retrouvé. Finalement, nous avons analysé par immunohistochimie les gonades dysgénésiques de 30 patients avec une anomalie du développement sexuel et un chromosome Y normal ou remanié. Nos travaux ont montré la présence de cellules germinales immatures au sein de cordons sexuels primitifs sous forme de tissu gonadique indifférencié dans 15 gonades, dont 9 ont évolué en tumeur gonadique. Dans 13 autres gonades, ces cellules germinales immatures avaient disparues par apoptose. Dans l'ensemble, notre recherche met en évidence la susceptibilité du chromosome Y à subir des remaniements et à être instable lors des divisions cellulaires, et indique que le mosaïcisme peut avoir des répercussions sur la détermination gonadique. Nos travaux montrent également que le tissu gonadique indifférencié peut évoluer vers deux entités, une tumeur gonadique ou une bandelette suite à l'apoptose des cellules germinales, mettant en lumière la nécessité d'analyser le tissu gonadique des patients XY avec dysgénésie gonadique dont les gonades sont laissées en place. / Sexual development is a complex process which depends on numerous genes, mutations possibly resulting in an abnormal sexual development. Furthermore, chromosome abnormalities can have important repercussions on gonadal determination, especially when it comes to the Y chromosome since it carries the master gene of male sexual development. First, we identified by molecular cytogenetics the breakpoint in 5 patients with an X;Y translocation and 10 patients with an isodicentric Y chromosome. We were thus able to show that some regions are more at risk of being rearranged, especially when they contain palindromes or other repeated sequences. We were also able to establish a relationship between the distance separating the centromere from the breakpoint and instability of isodicentric Y chromosomes during cell divisions. Second, we studied by cytogenetics the gonads of patients with a normal or rearranged Y chromosome and presenting an abnormal sexual development. We demonstrated loss of the rearranged Y chromosome in a majority of gonadal cells of the 10 analyzed patients, explaining their abnormal sexual phenotype. On the other hand, in 11 of the 12 patients with a normal Y chromosome, no mosaicism clearly explaining their abnormal gonadal determination was found. Finally, we also analyzed by immunohistochemistry the dysgenetic gonads of 30 patients with an abnormal sexual developement and a normal or rearranged Y chromosome. We showed the presence of immature germ cells in primitive sex cords as undifferentiated gonadal tissue in 15 gonads, including 9 that evolved in a gonadal tumor. In 13 other gonads, these immature germ cells had disappeared through apoptosis. Altogether, our research demonstrates that the Y chromosome is susceptible to rearrangements and can be unstable through cell divisions, and that mosaicism may have repercussions on gonadal determination. Our work also shows that undifferentiated gonadal tissue can evolve in two entities, a gonadal tumor or a streak following apoptosis of germ cells, thus emphasizing the necessity of studying the gonads of XY patients with gonadal dysgenesis when gonads are left in place.

Nos mamíferos, a determinação sexual é governada pelo equilíbrio entre duas vias de sinalização paralelas e antagônicas: a via masculina SOX9/FGF9 e a via feminina RSPO1/beta-catenina/WNT4. A R-spondina 1 é uma importante reguladora do processo de diferenciação ovariana e atua modulando a via de sinalização Wnt canônica (Wnt/beta-catenina). Em humanos, mutações em RSPO1 causam uma rara síndrome genética autossômica recessiva caracterizada por Distúrbios do Desenvolvimento Sexual (DDS) 46,XX Testicular ou Ovotesticular, hiperceratose palmoplantar (HPP) e predisposição para o desenvolvimento de carcinoma de células escamosas (MIM 610644). Identificamos um paciente brasileiro, proveniente de uma grande família consanguínea, que apresentava a associação de HPP e DDS 46,XX Testicular SRY negativo. A avaliação da região codificadora do gene RSPO1 identificou a nova variante alélica c.305G>A (p.Cys102Tyr). O estudo de segregação realizado em 67 familiares demonstrou que a variante c.305G>A segrega em perfeita concordância com o fenótipo de HPP, exibindo um padrão de herança autossômico recessivo. Na família foram identificados 10 indivíduos afetados pelo fenótipo de HPP. As avaliações clínica e hormonal e os estudos molecular e citogenético nesses indivíduos resultou na caracterização de: (a) quatro indivíduos do sexo masculino 46,XX e/ou SRY negativo, com ambiguidade genital e perfil hormonal alterado; (b) cinco indivíduos do sexo masculino 46,XY e/ou SRY positivo, sem ambiguidade genital, com perfil hormonal normal e (c) uma mulher 46,XX, fértil. Experimentos de transfecção transitória in vitro demostraram que a proteína mutante tem menor capacidade de transativação do plasmídio reporter da via Wnt. As simulações de dinâmica molecular constataram que a troca p.Cys102Tyr aumenta a flexibilidade do backbone da R-spondina-1, diminuindo a energia de ligação da proteína ao complexo de receptores, LGR5 e RNF43. Em conjunto, nossos achados demonstram que a variante c.305G > A é patogênica, sendo responsável pela síndrome genética diagnosticada na família brasileira. As análises de expressão gênica e os estudos de imuno-histoquímica, por sua vez, detectaram um aumento da expressão do gene SOX9 e maior imonorreatividade para a proteína Sox9 no tecido testicular do caso índice. Esses resultados sugerem que o processo de reversão sexual nos indivíduos XX ocorra por uma hiperexpressão de SOX9 secundária à menor ativação da via Wnt/beta-catenina na gônada durante a embriogênese. No presente estudo também relatamos o primeiro caso de indivíduo de cariótipo 46,XX portador de mutação em homozigose no gene RSPO1 que não desenvolveu DDS. A variabilidade do fenótipo sexual não está associada com alterações no número de cópias dos genes WNT4 ou do SOX9 e região cis-regulatória. No entanto, a avaliação do exoma da família encontrou uma associação entre o polimorfismo do receptor LGR5 rs17109924 e a atenuação do fenótipo de DDS. Todavia, serão necessários estudos funcionais para esclarecer o impacto biológico da interação das variantes RSPO1 p.Cys102Tyr e LGR5 rs171099 / In mammals, sex determination is governed by the balance between two parallel and antagonic signaling pathways: the male SOX9/FGF9 and the female, RSPO1/beta-catenin/WNT4 pathways. R-spondin 1 regulates the ovarian differentiation process by its modulating action through the canonic Wnt pathway (Wnt/beta-catenin). In humans, patogenic mutations in RSPO1 cause a rare, autosomic recessive syndrome characterized by 46,XX Testicular or Ovotesticular disorders of sexual development (DSD), palmoplantar keratosis (PPK) and predisposition to squamous cell carcinoma (MIM 610644). We identified and studied a SRY-negative 46,XX DSD patient with PPK from a large, consaguineous, brazillian family. Through a \"candidate gene\" approach we identified in the proband a new allelic variant in the coding region of RSPO1, c.305G > A. This variant presented full concordance with the PPK phenotype by segregation analyses in 10 of 67 members of this family. Clinical, hormonal, cytogenetic and molecular genetic studies characterized three patterns in individuals with this variant: (a) four 46,XX and/or SRY-negative males with ambiguous genitalia and altered hormonal profile; (b) five 46,XY and/or SRY-positive males without ambiguous genitalia with normal hormonal profile; (c) one 46,XX fertile woman. In vitro experiments demonstrated that transient transfection of the mutant protein resulted in lower transactivation of the Wnt pathway-reporter plasmid. Moreover, molecular dinamic studies showed that p.Cys102Tyr increased the R-spondin-1 backbone flexibility, thus decreasing the interaction between this protein and its receptors, LGR5 and RNF43. Thus, both in vitro and in silico analysis demonstrate the pathogenicity of the RSPO1 variant c.305G > A. In addition, in the index case, a higher expression of SOX9, corroborated by a reactive immunohistochemistry in testicular tissue, suggested that the process of sexual reversal in the XX individual is driven by a higher SOX9 expression possibly due to a lower Wnt/beta-catenin signaling pathway activation during embriogenesis. In this study, we also reported the first 46,XX individual with RSPO1 mutation without DSD, in which no copy number abnormality was detected in WNT4, SOX9 and its cisregulatory regions. Whole exome sequencing of the affected individuals revealed, in turn, that the LGR5 rs17109924 polymorphism associates with a protacted DSD phenotype in the fertile woman with normal hormonal profile. Despite this evidence, future studies are nedded to address causality and biological impact between RSPO1 p.Cys102Tyr and LGR5 rs17109924 variants

Le développement sexuel est un processus complexe qui dépend de nombreux gènes, une mutation pouvant entraîner un développement sexuel anormal. Par ailleurs, des anomalies chromosomiques peuvent avoir des répercussions importantes sur la détermination gonadique, surtout lorsqu'il s'agit du chromosome Y puisqu'il porte le gène clé du développement sexuel masculin. Premièrement, nous avons identifié par cytogénétique moléculaire le point de cassure chez 5 patients avec une translocation X;Y et 10 patients avec un chromosome Y isodicentrique. Nous avons ainsi démontré que certaines régions sont plus à risque d'être remaniées, notamment lorsqu'elles contiennent des palindromes ou d'autres séquences répétées. Nous avons également établi une relation entre la distance séparant le centromère et le point de cassure et l'instabilité des chromosomes Y isodicentriques lors des divisions cellulaires. Deuxièmement, nous avons étudié en cytogénétique les gonades de 22 patients avec un chromosome Y normal ou remanié et présentant un développement sexuel anormal. Nous avons mis en évidence la perte du chromosome Y remanié dans une majorité de cellules gonadiques des 10 patients étudiés, expliquant leur phénotype sexuel anormal. Cependant, chez 11 des 12 patients avec un chromosome Y normal, aucun mosaïcisme expliquant clairement leur détermination gonadique anormale n'a été retrouvé. Finalement, nous avons analysé par immunohistochimie les gonades dysgénésiques de 30 patients avec une anomalie du développement sexuel et un chromosome Y normal ou remanié. Nos travaux ont montré la présence de cellules germinales immatures au sein de cordons sexuels primitifs sous forme de tissu gonadique indifférencié dans 15 gonades, dont 9 ont évolué en tumeur gonadique. Dans 13 autres gonades, ces cellules germinales immatures avaient disparues par apoptose. Dans l'ensemble, notre recherche met en évidence la susceptibilité du chromosome Y à subir des remaniements et à être instable lors des divisions cellulaires, et indique que le mosaïcisme peut avoir des répercussions sur la détermination gonadique. Nos travaux montrent également que le tissu gonadique indifférencié peut évoluer vers deux entités, une tumeur gonadique ou une bandelette suite à l'apoptose des cellules germinales, mettant en lumière la nécessité d'analyser le tissu gonadique des patients XY avec dysgénésie gonadique dont les gonades sont laissées en place. / Sexual development is a complex process which depends on numerous genes, mutations possibly resulting in an abnormal sexual development. Furthermore, chromosome abnormalities can have important repercussions on gonadal determination, especially when it comes to the Y chromosome since it carries the master gene of male sexual development. First, we identified by molecular cytogenetics the breakpoint in 5 patients with an X;Y translocation and 10 patients with an isodicentric Y chromosome. We were thus able to show that some regions are more at risk of being rearranged, especially when they contain palindromes or other repeated sequences. We were also able to establish a relationship between the distance separating the centromere from the breakpoint and instability of isodicentric Y chromosomes during cell divisions. Second, we studied by cytogenetics the gonads of patients with a normal or rearranged Y chromosome and presenting an abnormal sexual development. We demonstrated loss of the rearranged Y chromosome in a majority of gonadal cells of the 10 analyzed patients, explaining their abnormal sexual phenotype. On the other hand, in 11 of the 12 patients with a normal Y chromosome, no mosaicism clearly explaining their abnormal gonadal determination was found. Finally, we also analyzed by immunohistochemistry the dysgenetic gonads of 30 patients with an abnormal sexual developement and a normal or rearranged Y chromosome. We showed the presence of immature germ cells in primitive sex cords as undifferentiated gonadal tissue in 15 gonads, including 9 that evolved in a gonadal tumor. In 13 other gonads, these immature germ cells had disappeared through apoptosis. Altogether, our research demonstrates that the Y chromosome is susceptible to rearrangements and can be unstable through cell divisions, and that mosaicism may have repercussions on gonadal determination. Our work also shows that undifferentiated gonadal tissue can evolve in two entities, a gonadal tumor or a streak following apoptosis of germ cells, thus emphasizing the necessity of studying the gonads of XY patients with gonadal dysgenesis when gonads are left in place.

Nos mamíferos, a determinação sexual é governada pelo equilíbrio entre duas vias de sinalização paralelas e antagônicas: a via masculina SOX9/FGF9 e a via feminina RSPO1/beta-catenina/WNT4. A R-spondina 1 é uma importante reguladora do processo de diferenciação ovariana e atua modulando a via de sinalização Wnt canônica (Wnt/beta-catenina). Em humanos, mutações em RSPO1 causam uma rara síndrome genética autossômica recessiva caracterizada por Distúrbios do Desenvolvimento Sexual (DDS) 46,XX Testicular ou Ovotesticular, hiperceratose palmoplantar (HPP) e predisposição para o desenvolvimento de carcinoma de células escamosas (MIM 610644). Identificamos um paciente brasileiro, proveniente de uma grande família consanguínea, que apresentava a associação de HPP e DDS 46,XX Testicular SRY negativo. A avaliação da região codificadora do gene RSPO1 identificou a nova variante alélica c.305G>A (p.Cys102Tyr). O estudo de segregação realizado em 67 familiares demonstrou que a variante c.305G>A segrega em perfeita concordância com o fenótipo de HPP, exibindo um padrão de herança autossômico recessivo. Na família foram identificados 10 indivíduos afetados pelo fenótipo de HPP. As avaliações clínica e hormonal e os estudos molecular e citogenético nesses indivíduos resultou na caracterização de: (a) quatro indivíduos do sexo masculino 46,XX e/ou SRY negativo, com ambiguidade genital e perfil hormonal alterado; (b) cinco indivíduos do sexo masculino 46,XY e/ou SRY positivo, sem ambiguidade genital, com perfil hormonal normal e (c) uma mulher 46,XX, fértil. Experimentos de transfecção transitória in vitro demostraram que a proteína mutante tem menor capacidade de transativação do plasmídio reporter da via Wnt. As simulações de dinâmica molecular constataram que a troca p.Cys102Tyr aumenta a flexibilidade do backbone da R-spondina-1, diminuindo a energia de ligação da proteína ao complexo de receptores, LGR5 e RNF43. Em conjunto, nossos achados demonstram que a variante c.305G > A é patogênica, sendo responsável pela síndrome genética diagnosticada na família brasileira. As análises de expressão gênica e os estudos de imuno-histoquímica, por sua vez, detectaram um aumento da expressão do gene SOX9 e maior imonorreatividade para a proteína Sox9 no tecido testicular do caso índice. Esses resultados sugerem que o processo de reversão sexual nos indivíduos XX ocorra por uma hiperexpressão de SOX9 secundária à menor ativação da via Wnt/beta-catenina na gônada durante a embriogênese. No presente estudo também relatamos o primeiro caso de indivíduo de cariótipo 46,XX portador de mutação em homozigose no gene RSPO1 que não desenvolveu DDS. A variabilidade do fenótipo sexual não está associada com alterações no número de cópias dos genes WNT4 ou do SOX9 e região cis-regulatória. No entanto, a avaliação do exoma da família encontrou uma associação entre o polimorfismo do receptor LGR5 rs17109924 e a atenuação do fenótipo de DDS. Todavia, serão necessários estudos funcionais para esclarecer o impacto biológico da interação das variantes RSPO1 p.Cys102Tyr e LGR5 rs171099 / In mammals, sex determination is governed by the balance between two parallel and antagonic signaling pathways: the male SOX9/FGF9 and the female, RSPO1/beta-catenin/WNT4 pathways. R-spondin 1 regulates the ovarian differentiation process by its modulating action through the canonic Wnt pathway (Wnt/beta-catenin). In humans, patogenic mutations in RSPO1 cause a rare, autosomic recessive syndrome characterized by 46,XX Testicular or Ovotesticular disorders of sexual development (DSD), palmoplantar keratosis (PPK) and predisposition to squamous cell carcinoma (MIM 610644). We identified and studied a SRY-negative 46,XX DSD patient with PPK from a large, consaguineous, brazillian family. Through a \"candidate gene\" approach we identified in the proband a new allelic variant in the coding region of RSPO1, c.305G > A. This variant presented full concordance with the PPK phenotype by segregation analyses in 10 of 67 members of this family. Clinical, hormonal, cytogenetic and molecular genetic studies characterized three patterns in individuals with this variant: (a) four 46,XX and/or SRY-negative males with ambiguous genitalia and altered hormonal profile; (b) five 46,XY and/or SRY-positive males without ambiguous genitalia with normal hormonal profile; (c) one 46,XX fertile woman. In vitro experiments demonstrated that transient transfection of the mutant protein resulted in lower transactivation of the Wnt pathway-reporter plasmid. Moreover, molecular dinamic studies showed that p.Cys102Tyr increased the R-spondin-1 backbone flexibility, thus decreasing the interaction between this protein and its receptors, LGR5 and RNF43. Thus, both in vitro and in silico analysis demonstrate the pathogenicity of the RSPO1 variant c.305G > A. In addition, in the index case, a higher expression of SOX9, corroborated by a reactive immunohistochemistry in testicular tissue, suggested that the process of sexual reversal in the XX individual is driven by a higher SOX9 expression possibly due to a lower Wnt/beta-catenin signaling pathway activation during embriogenesis. In this study, we also reported the first 46,XX individual with RSPO1 mutation without DSD, in which no copy number abnormality was detected in WNT4, SOX9 and its cisregulatory regions. Whole exome sequencing of the affected individuals revealed, in turn, that the LGR5 rs17109924 polymorphism associates with a protacted DSD phenotype in the fertile woman with normal hormonal profile. Despite this evidence, future studies are nedded to address causality and biological impact between RSPO1 p.Cys102Tyr and LGR5 rs17109924 variants