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Monogenic autoimmune diseases are highly variable syndromes that usually have onset in the first year of life and are often fatal in early childhood. Identifying monogenic autoimmune diabetes is important as it can have implications for medical management of patients, informs families and clinicians of prognosis and recurrence risk, and gives insights into beta-cell autoimmunity and immune tolerance. The first section of this thesis introduces monogenic autoimmune disease, with focus on the conditions that have autoimmune endocrine disorders as part of their clinical phenotype. The following section details the methodologies used throughout this thesis. In chapter 1, we used a type 1 diabetes genetic risk score (T1D-GRS) based on the top 10 risk alleles for T1D to identify patients with monogenic autoimmunity from patients with early-onset polygenic diabetes and additional autoimmunity. We showed that the T1D-GRS was highly discriminatory of monogenic autoimmunity, especially when combined with age of onset (ROC-AUC 0.88). We also identified 16 families for gene discovery studies. Furthermore, this work shows that polygenic risk for the development of T1D does not affect the development of diabetes in monogenic autoimmunity. Chapter 2 describes the genetic and phenotypic information for the largest cohort of patients with IPEX syndrome, caused by hemizygous mutations in FOXP3, reported to date (n=48). We analysed this data to determine if there were any genotypic or clinical characteristics of IPEX syndrome that could predict prognosis. We did not find evidence of phenotype-genotype relationships and showed that presenting feature did not predict prognosis. Medical management of IPEX syndrome cannot, therefore, be based on genotype or presentation. In chapter 3 we employed whole exome sequencing to look for causal variant(s) in a patient with diabetes (diagnosed aged 7 weeks) and autoimmune lymphoproliferative disease. This identified recessively inherited causative variants in LRBA. We then used targeted next generation sequencing (NGS) to screen a large cohort of patients (n=169) and identified an additional 8 probands and an affected family member. This confirms the role of LRBA as a neonatal diabetes gene, bringing the total number of genes to 25. In chapter 4, we assessed if immunoglobulin E (IgE) could be useful to identify patients with early-onset multisystem autoimmune disease caused by gain of function (GOF) STAT3 mutations. We showed that serum IgE was below the lower limit of the normal reference range (2KU/L) in all patients with STAT3 GOF (n=6), giving this threshold a sensitivity of 100% (95% CI: 54.1 – 100) and specificity 97.2% (95% CI: 96.2-97.9). We also found that IgE in patients with IPEX (n=16) was significantly higher than those with STAT3 GOF (p=0.002) suggesting it could be useful to identify IPEX from STAT3 GOF in non-consanguineous males with early-onset autoimmunity. The final concluding section summarises the key findings of each chapter, the impact of these findings and suggests future avenues for research. Identifying monogenic autoimmunity has enabled prenatal diagnoses, given families and clinicians knowledge on recurrence risk, and could enable targeted therapies to be employed. This body of work will enable better discrimination of monogenic autoimmunity from polygenic clustering of early-onset autoimmunity, and gives insights into the factors that determine disease phenotype and clinical course in monogenic autoimmunity. Gene discovery on the remaining patients will give new insights into the mechanisms of beta-cell autoimmunity and the regulation of the adaptive immune system and maintenance of immune tolerance.

La fonction endocrine du pancréas est essentielle pour l'homéostasie du glucose parce que les îlots pancréatiques contiennent le seul type des cellules endocrines, nommées cellules bêta, qui sont capable de produire et sécréter de l’insuline. Le facteur de transcription RFX6, maintenu dans toutes les cellules endocrines matures, est essentiel pour le développement, l'identité et la fonction des cellules bêta. Chez l'homme, des mutations de RFX6 causent le syndrome de Mitchell-Riley, un trouble du développement caractérisé par un diabète néonatal et des malformations du système gastro-intestinal. La recherche des cibles de RFX6 dans les îlots murins a révélé que le facteur de transcription Mlxipl est directement régulé par RFX6. Dans cette thèse, nous avons étudié le mécanisme de la régulation transcriptionnelle de Mlxipl par RFX6 ainsi que les rôles de RFX6 et MLXIPL dans les cellules bêta adultes. Nous avons démontré que RFX6 se lie au premier intron de Mlxipl qui contient un motif de liaison (xbox) critique, et nous avons identifié les cofacteurs de ce processus. En comparant l’effet de la répression de Rfx6 et Mlxipl dans des milieux riches ou faibles en glucose dans la lignée cellulaire bêta Ins-1 832/13 sur le transcriptome, nous avons déterminé les programmes génétiques contrôlés par RFX6 et MLXIPL. / Pancreatic endocrine function is critical for glucose homeostasis because pancreatic islets contain the only cells of the body, the beta cells, capable of producing and secreting insulin. The transcription factor RFX6 is maintained in all mature islet cells and is as an essential regulator of beta cell development, identity and function. In humans, RFX6 mutations cause Mitchell-Riley syndrome, a developmental disorder characterized by neonatal diabetes and malformations of the digestive tract. The search for RFX6 targets in murine islets revealed that the transcription factor Mlxipl is directly regulated by RFX6. In this thesis, we investigated the mechanism of Mlxipl transcriptional regulation by RFX6, and the respective roles of RFX6 and its downstream target MLXIPL in adult beta cells. We demonstrated that RFX6 binds to the first intron of Mlxipl that contains a critical RFX binding motif (xbox), and we identified cofactors of this process. By comparing the changes in the transcriptomes linked to the loss of RFX6 or MLXIPL in the pancreatic beta cell line Ins-1 832/13 and the glucose level, we determined the genetic programs controlled by RFX6 and MLXIPL.

Introdução: O diabetes monogênico corresponde de 1% a 2% de todos os casos de diabetes mellitus, sendo causado por variantes em um único gene. Dentre esses, o mais comum é o MODY (Maturity Onset Diabetes of the Young), havendo, porém, diversas formas mais raras, como diabetes neonatal e sindrômico que podem estar associadas a outras comorbidades além do diabetes, bem como às lipodistrofias hereditárias. O diagnóstico genético permite a adequação do tratamento, seguimento clínico e aconselhamento familiar. Objetivos: (1) desenvolver e implantar um painel customizado de sequenciamento em larga escala para o diagnóstico genético-molecular das formas raras de diabetes monogênico e lipodistrofias hereditárias; (2) estabelecer o diagnóstico genético de casos com suspeita clínica; (3) realizar a correlação genótipo-fenótipo. Métodos: Os pacientes foram selecionados de acordo com os critérios de inclusão para cada tipo de diabetes monogênico raro. A pesquisa genética dos casos-índice foi feita por sequenciamento em larga escala. A segregação familiar e confirmação dos achados foram feitas pelo método de Sanger. A análise de bioinformática foi realizada considerando o tipo de variante, frequência em bancos de dados populacionais, predição in silico e segregação familiar. Resultados: Foram analisados 42 casos, sendo que em 23 foram encontradas variantes candidatas: 6/16 com diagnóstico de diabetes neonatal, 2/2 com quadro clínico de Síndrome de Wolfram, 11/11 com suspeita de lipodistrofia congênita generalizada, 4/13 com lipodistrofia parcial familiar. Conclusões: Um painel customizado de sequenciamento em larga escala permitiu o diagnóstico genético-molecular de diabetes monogênico, com positividade de 22/42 casos analisados, sendo possível realizar a correlação genótipofenótipo. O diagnóstico genético possibilitou o aprimoramento do seguimento clínico dos pacientes e suas famílias. Permitiu, ainda, aumentar o número de diagnósticos de casos anteriormente subdiagnosticados / Introduction: Monogenic Diabetes accounts for 1 to 2% of all cases of diabetes mellitus. It is caused for variants in a single gene. Among these, the most common is MODY (Maturity Onset Diabetes of the Young), but there are several other rare forms, which may be associated with other comorbidities besides diabetes. Genetic diagnosis can lead to appropriate treatment and follow-up, besides and family counseling. Objectives: (1) to develop a customized targeted massively parallel sequencing panel to sequence the rare types of monogenic diabetes; (2) To establish the genetic diagnosis of probands with clinical suspicion of monogenic diabetes; (3) To correlate their phenotype with genetic findings, leading to a better understanding of rare monogenic diabetes subtypes. Methods: Patients were selected according to the inclusion criteria for each type of rare monogenic diabetes. Genetic research of index cases was done by massively parallel sequencing. Family segregation and confirmation of the variants findings were done by Sanger method. Bioinformatics analysis was performed considering the type of variant, frequency in population databases, in silico prediction and family segregation. Results: We analyzed a total of 42 cases. We found 22 candidate causal variants: 6/16 in probands with Neonatal Diabetes, 2/2 with Wolfram Syndrome, 11/11 with suspected Generalized Congenital Lipodystrophy, and 4/13 with Familial Partial Lipodystrophy. Conclusions: A customized targeted massively parallel sequencing panel allowed genetic diagnosis of rare types of monogenic diabetes, with a positivity of 23/42 cases analyzed, being possible to perform the genotype-phenotype correlation. The genetic diagnosis allowed the improvement of the clinical follow-up of the patients and their families. It also increased the number of diagnoses of previously underdiagnosed cases

A diabetes é um distúrbio endócrino do metabolismo dos carboidratos, clinicamente caracterizada por hiperglicemia, resultante da incapacidade do organismo em secretar insulina, defeitos na sua ação ou ambos. Na última década, houve um crescente aumento no número de trabalhos sobre a múltipla hereditariedade de um tipo de diabetes rara e não imunológica diagnosticada antes dos 6 meses de vida, a diabetes neonatal (DN). A maioria dos estudos, existentes na literatura referentes à DN, foi realizada em pacientes e aborda principalmente aspectos clínicos, etiológicos e terapêuticos. No entanto, existe uma deficiência de estudos realizados em modelos animais, a fim de avaliar danos moleculares em tecidos submetidos à hiperglicemia neonatal. Recentemente, as consequências da diabetes no sistema nervoso central (SNC) têm recebido maior atenção, uma vez que os recentes estudos mostram que a hiperglicemia é capaz de promover a ruptura da homeostase redox no cérebro de ratos. Neste sentido, o estresse oxidativo (EO) parece representar um dos mecanismos pelos quais a hiperglicemia danifica o tecido cerebral em um período crucial de desenvolvimento. Diante disso, o presente trabalho objetivou estudar não só a relação do EO com a hiperglicemia neonatal em cérebro de ratos, mas também avaliar se os danos oxidativos promovidos pelas espécies reativas de oxigênio (ERO) na condição hiperglicêmica podem estar envolvidos no processo de morte celular neuronal. Para isso, foram utilizados ratos Wistar de 5 dias de vida, divididos em dois grupos: controle e diabético. O modelo de diabetes foi induzido pela administração intraperitoneal de estreptozotocina (STZ) em uma única dose de 100 mg/Kg peso corporal, sendo que foram considerados diabéticos os ratos com glicemia >200mg/dL. Os animais foram sacrificados com 10 dias de vida, ou seja, 5 dias após a administração de STZ. O cérebro total dos animais foi homogeneizado, centrifugado e o homogeneizado utilizado para as medidas de parâmetros de EO e expressão proteica. Além disso, o cérebro total foi utilizado em cortes histológicos para análise do parâmetro de morte celular neuronal, avaliada pela técnica FluoroJade C. Os parâmetros de EO analisados foram o metabolismo da glutationa, que engloba a atividade das enzimas glutationa S-transferase (GST), glutationa redutase (GR), glutamato-cisteína ligase (GCL) e a 8 determinação da concentração de glutationa total e reduzida (GSH/GSSG). A medida do peróxido de hidrogênio (H2O2) também foi avaliada, juntamente com a quantificação proteica por “Western Blot” do fator nuclear eritroide relacionado ao fator 2 (Nrf2), da superóxido dismutase (SOD), da catalase (CAT), da glutationa peroxidase (GPx), da heme oxigenase 1 (HO-1) e da tiorredoxina (TRX). Os parâmetros relativos à sobrevivência e morte celular avaliados foram a quantificação proteica por “Western Blot” da proteína cinase B (AKT), da proteína cinase B fosforilada (p-AKT), da glicogênio sintase cinase 3 β (GSK3β), da p38 proteína cinase ativada por mitógenos (p38), proteína cinase c-Jun N-terminal (JNK), da célula-B de linfoma 2 (Bcl2) e da proteína X associada a Bcl2 (Bax). Os ratos submetidos ao modelo de hiperglicemia neonatal não apresentaram diferenças significativas nos parâmetros relacionados ao metabolismo da glutationa (GST, GR, GCL e GSH/GSSG), tampouco nas concentrações de H2O2 quando comparados ao grupo controle. A expressão proteica do Nrf2 foi diminuída no grupo diabético, enquanto que a expressão da CAT, HO-1 e TRX se apresentaram aumentadas no grupo diabético quando comparado ao grupo controle. Não foram encontradas diferenças significativas nas expressões proteicas da SOD e GPx. As expressões proteicas das proteínas p38 e Bcl2 foram aumentadas, enquanto a expressão da p-AKT se mostrou reduzida no grupo diabético. Já com relação à expressão das proteínas JNK, GSK3β e Bax não houve diferença significativa nos grupos analisados. Finalmente, com relação à técnica que avaliou morte celular neuronal, o grupo diabético apresentou três vezes mais marcações de neurônios fluorescentes, ou seja, com morte celular quando comparado com o grupo controle. Portanto, esses resultados sugerem que o EO pode representar um mecanismo envolvido nos efeitos da hiperglicemia no SNC de ratos neonatos. Além disso, as alterações na expressão de proteínas envolvidas em vias de sobrevivência/morte celular colaboram para o resultado de morte celular verificado no cérebro de animais com hiperglicemia neonatal e mostram os efeitos nocivos da DN em um período crucial de desenvolvimento cerebral. / Diabetes is an endocrine disorder of the carbohydrates metabolism clinically characterized by hyperglycemia, resulting from the inability of the body to secrete insulin, defeats in its action and both. In the last decade, there has been an increasing number of studies about neonatal diabetes (DN), a type of diabetes non-immunological diagnosed before 6 months of life. The most studies related to DN was developed in patients and mainly deal with clinical, etiological and therapeutic aspects. However, there is a few of studies in animal models in order to assess molecular damage in tissues submitted to neonatal hyperglycemia. Recently, the consequences of diabetes in the central nervous system (CNS) have received increased attention, as recent studies show that hyperglycemia is capable of promoting the rupture of redox homeostasis in the rat brain. Wherefore, the present work aimed to study not only the relationship between EO and neonatal hyperglycemia in rat brain, but also evaluate if the oxidative damage promoted by reactive oxygen species (ROS) in the hyperglycemic condition may be involved in the neuronal cell death process. For this, 5-day-old Wistar rats were used to promote the induction of diabetes, which was done with a single intraperitoneal streptozotocin (STZ) administration (100 mg/kg body weight). Rats with glycemia> 200 mg/dL were considered diabetic. The rats were sacrificed in 10 days of life, wherefore five days after STZ adiminstration. The whole brain of the rats was homogenized, centrifuged and homogenized used for EO techniques and protein expression measurements. In addition, total brain was used in histological sections for analysis of the neuronal cell death. The EO parameters evaluated were the activity of the glutathione S-transferase (GST), glutathione reductase (GR), glutamate-cystein ligase (GCL) and the determination of total and reduced glutathione concentration (GSH/GSSG). Hydrogen peroxide (H2O2) was evaluated along with Western Blot protein quantification of catalase (CAT), glutathione peroxidase (GPx), heme oxygenase (HO-1) and thioredoxin (TRX). Relative to cell survival and death were evaluated protein kinase B (Akt), phosphorylated protein kinase B (p-Akt), glycogen synthase kinase 3β (GSK3β), p38 mitogen-activated protein kinase (p38), c-Jun amino-terminal kinases (JNK), phosphorylated c-Jun amino-terminal kinases (p-JNK), B-cell 10 lymphoma 2 (Bcl2) and Bcl2-associated protein X (Bax) by western blot. The neonatal hyperglycemia was not able to promote significant differences in the glutathione metabolism (GST, GR, GCL and GSH / GSSG) nor in the H2O2 measurement when compared to the control group. Nrf2 protein expression was decreased whereas CAT, HO-1 and TRX protein expression were increased in the diabetic group when compared to the control group. No significant differences were found in the protein expression of SOD and GPx. Also, p38 and Bcl2 protein expression was increased, whereas p-Akt was decreased in the diabetic group, already regarding the expression of JNK, p-JNK, Jsk3β and Bax proteins there were no significant difference in the analyzed groups. Finally, relative to neuronal cell death technique, the diabetic group presented three fold more neuronal cell death with fluorescent marking characteristic, when compared to the control group. Therefore, these results suggest that OE may represent a mechanism involved in the effects of hyperglycemia in the central nervous system of neonatal rats. In addition, changes in the expression of proteins involved in survival/death cell pathways contribute to the outcome of cell death, result found in the brain of animals with neonatal hyperglycemia and finally show the harmful effects of neonatal diabetes in a crucial period of brain development.

A diabetes é um distúrbio endócrino do metabolismo dos carboidratos, clinicamente caracterizada por hiperglicemia, resultante da incapacidade do organismo em secretar insulina, defeitos na sua ação ou ambos. Na última década, houve um crescente aumento no número de trabalhos sobre a múltipla hereditariedade de um tipo de diabetes rara e não imunológica diagnosticada antes dos 6 meses de vida, a diabetes neonatal (DN). A maioria dos estudos, existentes na literatura referentes à DN, foi realizada em pacientes e aborda principalmente aspectos clínicos, etiológicos e terapêuticos. No entanto, existe uma deficiência de estudos realizados em modelos animais, a fim de avaliar danos moleculares em tecidos submetidos à hiperglicemia neonatal. Recentemente, as consequências da diabetes no sistema nervoso central (SNC) têm recebido maior atenção, uma vez que os recentes estudos mostram que a hiperglicemia é capaz de promover a ruptura da homeostase redox no cérebro de ratos. Neste sentido, o estresse oxidativo (EO) parece representar um dos mecanismos pelos quais a hiperglicemia danifica o tecido cerebral em um período crucial de desenvolvimento. Diante disso, o presente trabalho objetivou estudar não só a relação do EO com a hiperglicemia neonatal em cérebro de ratos, mas também avaliar se os danos oxidativos promovidos pelas espécies reativas de oxigênio (ERO) na condição hiperglicêmica podem estar envolvidos no processo de morte celular neuronal. Para isso, foram utilizados ratos Wistar de 5 dias de vida, divididos em dois grupos: controle e diabético. O modelo de diabetes foi induzido pela administração intraperitoneal de estreptozotocina (STZ) em uma única dose de 100 mg/Kg peso corporal, sendo que foram considerados diabéticos os ratos com glicemia >200mg/dL. Os animais foram sacrificados com 10 dias de vida, ou seja, 5 dias após a administração de STZ. O cérebro total dos animais foi homogeneizado, centrifugado e o homogeneizado utilizado para as medidas de parâmetros de EO e expressão proteica. Além disso, o cérebro total foi utilizado em cortes histológicos para análise do parâmetro de morte celular neuronal, avaliada pela técnica FluoroJade C. Os parâmetros de EO analisados foram o metabolismo da glutationa, que engloba a atividade das enzimas glutationa S-transferase (GST), glutationa redutase (GR), glutamato-cisteína ligase (GCL) e a 8 determinação da concentração de glutationa total e reduzida (GSH/GSSG). A medida do peróxido de hidrogênio (H2O2) também foi avaliada, juntamente com a quantificação proteica por “Western Blot” do fator nuclear eritroide relacionado ao fator 2 (Nrf2), da superóxido dismutase (SOD), da catalase (CAT), da glutationa peroxidase (GPx), da heme oxigenase 1 (HO-1) e da tiorredoxina (TRX). Os parâmetros relativos à sobrevivência e morte celular avaliados foram a quantificação proteica por “Western Blot” da proteína cinase B (AKT), da proteína cinase B fosforilada (p-AKT), da glicogênio sintase cinase 3 β (GSK3β), da p38 proteína cinase ativada por mitógenos (p38), proteína cinase c-Jun N-terminal (JNK), da célula-B de linfoma 2 (Bcl2) e da proteína X associada a Bcl2 (Bax). Os ratos submetidos ao modelo de hiperglicemia neonatal não apresentaram diferenças significativas nos parâmetros relacionados ao metabolismo da glutationa (GST, GR, GCL e GSH/GSSG), tampouco nas concentrações de H2O2 quando comparados ao grupo controle. A expressão proteica do Nrf2 foi diminuída no grupo diabético, enquanto que a expressão da CAT, HO-1 e TRX se apresentaram aumentadas no grupo diabético quando comparado ao grupo controle. Não foram encontradas diferenças significativas nas expressões proteicas da SOD e GPx. As expressões proteicas das proteínas p38 e Bcl2 foram aumentadas, enquanto a expressão da p-AKT se mostrou reduzida no grupo diabético. Já com relação à expressão das proteínas JNK, GSK3β e Bax não houve diferença significativa nos grupos analisados. Finalmente, com relação à técnica que avaliou morte celular neuronal, o grupo diabético apresentou três vezes mais marcações de neurônios fluorescentes, ou seja, com morte celular quando comparado com o grupo controle. Portanto, esses resultados sugerem que o EO pode representar um mecanismo envolvido nos efeitos da hiperglicemia no SNC de ratos neonatos. Além disso, as alterações na expressão de proteínas envolvidas em vias de sobrevivência/morte celular colaboram para o resultado de morte celular verificado no cérebro de animais com hiperglicemia neonatal e mostram os efeitos nocivos da DN em um período crucial de desenvolvimento cerebral. / Diabetes is an endocrine disorder of the carbohydrates metabolism clinically characterized by hyperglycemia, resulting from the inability of the body to secrete insulin, defeats in its action and both. In the last decade, there has been an increasing number of studies about neonatal diabetes (DN), a type of diabetes non-immunological diagnosed before 6 months of life. The most studies related to DN was developed in patients and mainly deal with clinical, etiological and therapeutic aspects. However, there is a few of studies in animal models in order to assess molecular damage in tissues submitted to neonatal hyperglycemia. Recently, the consequences of diabetes in the central nervous system (CNS) have received increased attention, as recent studies show that hyperglycemia is capable of promoting the rupture of redox homeostasis in the rat brain. Wherefore, the present work aimed to study not only the relationship between EO and neonatal hyperglycemia in rat brain, but also evaluate if the oxidative damage promoted by reactive oxygen species (ROS) in the hyperglycemic condition may be involved in the neuronal cell death process. For this, 5-day-old Wistar rats were used to promote the induction of diabetes, which was done with a single intraperitoneal streptozotocin (STZ) administration (100 mg/kg body weight). Rats with glycemia> 200 mg/dL were considered diabetic. The rats were sacrificed in 10 days of life, wherefore five days after STZ adiminstration. The whole brain of the rats was homogenized, centrifuged and homogenized used for EO techniques and protein expression measurements. In addition, total brain was used in histological sections for analysis of the neuronal cell death. The EO parameters evaluated were the activity of the glutathione S-transferase (GST), glutathione reductase (GR), glutamate-cystein ligase (GCL) and the determination of total and reduced glutathione concentration (GSH/GSSG). Hydrogen peroxide (H2O2) was evaluated along with Western Blot protein quantification of catalase (CAT), glutathione peroxidase (GPx), heme oxygenase (HO-1) and thioredoxin (TRX). Relative to cell survival and death were evaluated protein kinase B (Akt), phosphorylated protein kinase B (p-Akt), glycogen synthase kinase 3β (GSK3β), p38 mitogen-activated protein kinase (p38), c-Jun amino-terminal kinases (JNK), phosphorylated c-Jun amino-terminal kinases (p-JNK), B-cell 10 lymphoma 2 (Bcl2) and Bcl2-associated protein X (Bax) by western blot. The neonatal hyperglycemia was not able to promote significant differences in the glutathione metabolism (GST, GR, GCL and GSH / GSSG) nor in the H2O2 measurement when compared to the control group. Nrf2 protein expression was decreased whereas CAT, HO-1 and TRX protein expression were increased in the diabetic group when compared to the control group. No significant differences were found in the protein expression of SOD and GPx. Also, p38 and Bcl2 protein expression was increased, whereas p-Akt was decreased in the diabetic group, already regarding the expression of JNK, p-JNK, Jsk3β and Bax proteins there were no significant difference in the analyzed groups. Finally, relative to neuronal cell death technique, the diabetic group presented three fold more neuronal cell death with fluorescent marking characteristic, when compared to the control group. Therefore, these results suggest that OE may represent a mechanism involved in the effects of hyperglycemia in the central nervous system of neonatal rats. In addition, changes in the expression of proteins involved in survival/death cell pathways contribute to the outcome of cell death, result found in the brain of animals with neonatal hyperglycemia and finally show the harmful effects of neonatal diabetes in a crucial period of brain development.

No description available.