Congenital Adrenal Hyperplasia (CAH) describes a set of autosomal recessive diseases which affect enzymes mediating steroid biochemistry in the adrenal glands. In chromosomal females, the excess androgens associated with CAH cause virilization. Females with a high degree of virilisation can undergo feminizing genitoplasty in infancy or later in life. Parents and guardians are the medical proxies for their infants and therefore make decisions on their behalf. However, decision-making about feminizing genitoplasty can be very difficult. One tool that could help in such a situation is an electronic patient decision aid (PtDA). However, a PtDA for feminizing genitoplasty does not exist and there is insufficient information in existing literature to inform its design and development. Thus, the objectives of this study were to: (1) Identify user requirements, (2) Develop specifications for the design and development of the PtDA, and (3) Understand the best way to implement and distribute the PtDA We used the persona-scenario methodology to acquire user-requirements. Persona-scenario sessions were conducted with four parents of children with CAH, two adult patients with CAH, and four healthcare practitioners. Participants created fictitious personas, and scenarios wherein their personas interacted with an idealized version of the PtDA. Transcripts of these persona-scenarios and facilitator notes were analyzed to identify user-requirements, which were interpreted into specifications. Participants provided user requirements about (1) information and decisional content in the PtDA, (2) proposed functionalities for the PtDA, (3) web usability, and (4) implementation context. Many of these requirements are supported by existing literature. The requirements identified in this project will inform the design and development of a PtDA for feminizing genitoplasty in patients with CAH. However, further research is necessary to understand how to best implement these requirements and to ensure that the gathered information is useful for a broad range of potential end users. / Thesis / Master of Science (MSc)

Introduction: Sodium chloride supplementation in saltwasting congenital adrenal hyperplasia (CAH) is generally recommended in infants, but its implementation in routine care is very heterogeneous. Objective: To evaluate oral sodium chloride supplementation, growth, and hydrocortisone and fludrocortisone dose in infants with salt-wasting CAH due to 21-hydroxylase in 311 infants from the AQUAPE CAH database. Results: Of 358 patients with classic CAH born between 1999 and 2015, 311 patients had salt-wasting CAH (133 females, 178 males). Of these, 86 patients (27.7%) received oral sodium chloride supplementation in a mean dose of 0.9 ± 1.4 mmol/kg/day (excluding nutritional sodium content) during the first year of life. 225 patients (72.3%) were not treated with sodium chloride. The percentage of sodium chloride-supplemented patients rose from 15.2% in children born 1999–2004 to 37.5% in children born 2011–2015. Sodium chloride-supplemented and -unsupplemented infants did not significantly differ in hydrocortisone and fludrocortisone dose, target height-corrected height-SDS, and BMI-SDS during the first 2 years of life. Conclusion: In the AQUAPE CAH database, approximately one-third of infants with salt-wasting CAH receive sodium chloride supplementation. Sodium chloride supplementation is performed more frequently in recent years. However, salt supplementation had no influence on growth, daily fludrocortisone and hydrocortisone dose, and frequency of adrenal crisis.

A hiperplasia adrenocortical macronodular primária (PMAH) é uma causa rara de Síndrome de Cushing (SC). Apresenta como características macronódulos funcionantes em ambas as glândulas suprarrenais e uma produção variável de cortisol. Os nódulos apresentam uma produção ectópica de hormônio adrenocorticotrófico (ACTH) tornando a doença independente do ACTH hipofisário. Estudos independentes mostraram que mutações germinativas no gene armadillo repeat cointaining 5 (ARMC5) são uma causa frequente de PMAH. Além dessa mutação parece haver a participação de receptores hormonais ectópicos no córtex suprarrenal, que estimulariam a esteroidogênese e a hiperplasia da glândula. No entanto, os estudos sobre a relação entre o ARMC5 e a participação dos receptores aberrantes na produção de cortisol são incipientes. Portanto, temos como hipótese que as alterações no gene ARMC5 podem estar envolvidas no padrão celular e funcional das células que compõem os nódulos hiperplásicos na PMAH. Desta forma, nossos objetivos foram analisar: 1) a proporção de células espongiformes e compactas em cortes corados com hematoxilina e eosina; 2) a expressão gênica, nas células espongiformes e proteica de ambas as células, do ARMC5, das enzimas esteroidogênicas StAR, 3βHSD2 e CYP17A1, dos receptores da vasopressina (AVP1AR), serotonina (5HT4R) e do peptídeo inibidor gástrico (GIPR) e do ACTH ectópico e 3) a expressão do antígeno de proliferação celular, a proteína PCNA, para análise do padrão de proliferação em cortes histológicos de nódulos de hiperplasias de pacientes que apresentam mutações germinativas, mutações germinativas e somáticas ou não mutação no gene ARMC5. Os resultados mostraram que os nódulos hiperplásicos são compostos, na sua maioria, por células espongiformes, cujo padrão foi independente da presença de mutação. A expressão do ARMC5 nas células espongiformes foi maior em pacientes com mutação, e os receptores ectópicos apresentaram uma expressão maior no tecido de PMAH em relação à adrenal normal. A reação de imunoistoquimica revelou células positivas para a proteína ARMC5 e StAR, em ambos os tipos celulares, enquanto a enzima 3βHSD2 foi predominante nas células espongiformes e a enzima CYP17A1 nas células compactas. A expressão de AVP1AR, 5HT4R, e do ACTH ectópico foi positiva em ambos os tipos celulares e independente da presença ou não da mutação no gene ARMC5, bem como a expressão da proteína PCNA. Portanto, nossos resultados sugerem que a presença ou não de mutações no gene ARMC5 nas hiperplasias não está envolvida no padrão celular das células que compõem os nódulos. O mesmo para a presença dos receptores AVP1AR, 5HT4R e GIPR, do ACTH ectópico e da proteína PCNA. Como conclusão, os resultados dos parâmetros analisados sugerem que as alterações no gene ARMC5 não estão envolvidas no padrão celular e funcional das células que compõem os nódulos hiperplásicos na PMAH, e podem ter o mesmo grau de importância na formação desses nódulos. / The primary macronodular adrenal hyperplasia (PMAH) is a rare cause of Cushing\'s Syndrome (SC). It is characterized as macronodules in the adrenal gland and by variable production of cortisol. The nodules present an ectopic production of adrenocorticotrophic hormone (ACTH) pituitary-independent. Several studies showed that the germ mutations in armadillo repeat cointaining 5 (ARMC5) are a frequent cause of PMAH. In addition, there is a participation of ectopic hormonal receptors in the adrenal cortex, which promotes steroidogenesis and hyperplasia of the gland. However, studies on a relationship between ARMC5 and aberrant receptor involvement in cortisol production are incipient. Therefore, our hypothesis is that ARMC5 gene may be involved in the cellular and functional pattern of the cells in PMAH. Thus, the objectives were to analyzed: 1) the proportion of spongiform and compact cells in sections stained with hematoxylin and eosin; 2) the gene expression in the spongiform cells and the gene and protein expression in both cells types of the ARMC5, the steroidogenic enzymes StAR ,3βHSD2 e CYP17A1, and the ectopic receptors of vasopressin (AVP1AR), serotonin (5HT4R) and gastric inhibitory peptide (GIPR) also the ACTH ectopic and 3) the proliferating cell nuclear antigen (PCNA) that shows the proliferation pattern in patients PMAH that present germline, germinative and somatic mutations or no mutation in ARMC5 gene. We showed that the hyperplastic nodules are mainly composed by spongiform cells independent of the ARMC5 mutation. The expression of ARMC5 in spongiform cells was higher in patients with mutation, and PMAH tissue have a higher expression of the ectopic receptors when compared with normal adrenal. With the immunohistochemical labeling, we observed the ARMC5 and StAR protein expression in both cell types. The 3βHSD2 enzyme was predominant in spongiform cells and the CYP17A1 enzyme in the compact cells. The expression of AVP1AR, 5HT4R, and ectopic ACTH was positive in both cell types regardless of the presence of the ARMC5 mutation, as well as the expression of the PCNA protein. Therefore, the results suggest that the mutation in ARMC5 is not involved with spongiform and compact cell function. Moreover, the presence of the AVP1AR, 5HT4R and GIPR receptors, the ectopic ACTH and the PCNA protein was not related to the gene mutation. In conclusion, the alterations in the ARMC5 gene are not involved in the cell functional pattern in the PMAH and in the onset of the nodules.

A hiperplasia adrenal congênita (HAC) por deficiência da enzima 21-hidroxilase (21OH) é uma doença autossômica recessiva que compromete a síntese de cortisol e/ou aldosterona. É a causa mais frequente de distúrbio da diferenciação sexual 46,XX. Apresenta uma diversidade fenotípica, a qual é decorrente de mutações no gene CYP21A2. Observa-se forte correlação do comprometimento da atividade enzimática predita pelo genótipo com a forma de apresentação clínica e com os valores hormonais; entretanto, esta correlação não é observada com o grau de virilização pré-natal da genitália externa em mulheres com a forma clássica. Supomos que variações inter-individuais na ação, síntese e metabolismo dos andrógenos possam influenciar o fenótipo da virilização. Objetivos: avaliar se variantes alélicas em genes relacionados a ação, síntese ou metabolismo de andrógenos na vida fetal possuem um efeito modulatório na variabilidade fenotípica da virilização genital em mulheres com a forma clássica carreando genótipos 21OH semelhantes. Também serão avaliadas se diferenças na expressão tecidual local dos genes HSD17B5, SRD5A1, SRD5A2 e RA influenciariam esta variabilidade fenotípica da forma clássica. Casuística: foram selecionadas 187 mulheres com a forma clássica da HAC 21OH provenientes de 4 centros médicos. Dados clínicos, hormonais e o grau de virilização genital foram obtidos de forma retrospectiva da análise de prontuários. A intensidade de virilização genital foi classificada de acordo com a escala de Prader (P) e as pacientes foram divididas em 4 grupos: P I+II, P III, P IV e P V. As pacientes também foram agrupadas de acordo com o genótipo 21OH: grupo A carreadoras de mutações que predizem < 2% de atividade enzimática residual (n= 122) e grupo B carreadoras de mutações que predizem 3 a 7% de atividade residual (n= 58). Metodologias: foram amplificadas e re-sequenciadas as regiões que flanqueiam os exons dos genes CYP3A7, PXR e CAR. Para as variantes funcionais, foram re-sequenciados os exons 12-13 do POR e a região promotora do HSD17B5. As variantes V89L e A49T do SRD5A2 foram rastreadas por PCR-RFLP e o nCAG do RA por eletroforese capilar e análise pelo GeneScan. A determinação da expressão gênica em pele genital foi feita por PCR em tempo real utilizando os genes endógenos CYC, PGK1 e B2M. Os testes t-test, Mann-Whitney, Kruskal-Wallis, Fisher e regressão linear uni- e múltipla foram utilizados na análise estatística. Resultados: o Prader score no genótipo A variou de II a V (III: III IV) e no genótipo B de I a V (III: II - III) (P< 0,001). Foram encontradas em 2,5% dos alelos a variante CYP3A7*1C, em 24% CYP3A7*2, 31% rs2307424 CAR, 25% A503V POR, 33% -71G HSD17B5, 17% rs2518047 HSD17B5, 31% V89L SRD5A2 e em 1% dos alelos a variante A49T SRD5A2. Foi identificada associação das variantes rs2307424 CAR (P= 0,023 ;r2= 0,253) e rs2518047 HSD17B5 (P= 0,006; r2= 0,144) com o grau de virilização genital, tem sido encontradas em maior frequência no grupo de pacientes com virilização mais intensa. Todas as outras variantes não apresentaram associação com o Prader score (P> 0,05). As diferenças de expressão de todos os genes analisados em amostras de pele genital não foram estatisticamente significantes (P> 0,05), embora observou-se que em 4/7 amostras de pacientes com Prader score IV houve uma super-expressão do gene SRD5A2 em relação a 1/5 pacientes com Prader score III. Conclusão: neste estudo multicêntrico observamos que o genótipo 21OH se correlacionou com a intensidade de virilização genital em mulheres com a forma clássica. A variante rs2307424 do gene CAR, relacionada ao metabolismo pré-natal de andrógenos, e a variante rs2518047 do gene HSD17B5, relacionada à síntese de testosterona, associaram-se à fenótipos de virilização mais intensos. Não identificamos diferenças na expressão tecidual dos genes relacionados à síntese e/ou ação periférica de andrógenos em pacientes com os diferentes graus de virilização / Congenital Adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OH) is an autosomic recessive disorder characterized by an impairment in the cortisol and/or aldosterone synthesis, being the most frequent cause of 46,XX disorder of sex development. The disease presents a wide phenotypic variability resulting from different CYP21A2 gene mutations and a strong correlation has been observed among genotypes, clinical forms and basal hormone levels. However, this correlation is not observed regarding the degree of prenatal external genitalia virilization in females and an interindividual variability in the synthesis, metabolism and/or peripheral action of androgens could corroborate for these findings. Objectives: to evaluate if allelic variants in genes related to the androgen synthesis, metabolism and peripheral action could modulate the genital phenotype in CAH females bearing similar CYP21A2 mutations. Differences in the HSD17B5, SRD5A1, SRD5A2 and RA gene expression in genital skin were evaluated among patients with different degrees of external genital virilization. Patients: were selected 187 CAH females and clinical and hormonal data were retrospectively evaluated. The degree of external genitalia virilization was classified according to Prader (P) scores and patients were divided into 4 groups: P I+II, P III, P IV and P V. Patients were also grouped according to 21OH genotypes: group A bearing mutations predicting < 2% of residual enzymatic activity (n= 122) and group B between 3 to 7% (n= 58). Methodology: the exonic flanking regions of CYP3A7, PXR e CAR genes were PCR amplified and sequenced. The exons 12-13 of POR and the promotor region of HSD17B5 were sequenced to screen the functional polymorphisms. The V89L and A49T SRD5A2 alleles were screened by PCR-RFLP and the CAG polymorphic tract of AR gene by capillary electrophoresis and GeneScan analysis. The differential gene expression in genital skin was evaluated by real time PCR and the CYC, PGK1 e B2M housekeeping genes were used. The t-test, Mann-Whitney, Kruskal-Wallis, Fisher and uni- and multiple linear regression tests were used in statistical analysis. Results: Prader score in group A varied from II to V (III: III - IV) and in group B from I to V (III: II - III) (P< 0,001). The CYP3A7*1C allele was identified in 2.5% of alleles, CYP3A7*2 in 24%, rs2307424 CAR in 31%, A503V POR in 25%, -71G HSD17B5 in 33%, rs2518047 HSD17B5 in 17%, V89L SRD5A2 em 31% and A49T SRD5A2 in 1% of alleles. The rs2307424 CAR (P= 0.023; r2= 0.253) and rs2518047 HSD17B5 variants (P= 0.006; r2= 0.144) were associated with the degree of external genitalia virilization, and they were found in a higher frequency in more virilized patients. The remaining variants were not associated with Prader scores (P> 0.05). The HSD17B5, SRD5A1, SRD5A2 and RA gene expressions did not significantly differ between patients presenting Prader score III and IV (P> 0.05); however, 4/7 samples from patients with Prader IV and just 1/5 patients with Prader III presented an increased SRD5A2 expression. Conclusion: In this multicentric study the 21OH genotypes were correlated with the degree of external genitalia virilization in CAH females. The rs2307424 CAR and the rs2518047 HSD17B5 variants, related to the prenatal androgen metabolism and synthesis, respectively, explained some of the interindividual variability of genital phenotype in CAH females bearing similar CYP21A2 mutations. Differences in the expression of genes involved in the peripheral androgen action did not corroborate for the variability of genital phenotype in CAH

A hiperplasia adrenal congênita (HAC) por deficiência da enzima 21-hidroxilase (21OH) é uma doença autossômica recessiva que compromete a síntese de cortisol e/ou aldosterona. É a causa mais frequente de distúrbio da diferenciação sexual 46,XX. Apresenta uma diversidade fenotípica, a qual é decorrente de mutações no gene CYP21A2. Observa-se forte correlação do comprometimento da atividade enzimática predita pelo genótipo com a forma de apresentação clínica e com os valores hormonais; entretanto, esta correlação não é observada com o grau de virilização pré-natal da genitália externa em mulheres com a forma clássica. Supomos que variações inter-individuais na ação, síntese e metabolismo dos andrógenos possam influenciar o fenótipo da virilização. Objetivos: avaliar se variantes alélicas em genes relacionados a ação, síntese ou metabolismo de andrógenos na vida fetal possuem um efeito modulatório na variabilidade fenotípica da virilização genital em mulheres com a forma clássica carreando genótipos 21OH semelhantes. Também serão avaliadas se diferenças na expressão tecidual local dos genes HSD17B5, SRD5A1, SRD5A2 e RA influenciariam esta variabilidade fenotípica da forma clássica. Casuística: foram selecionadas 187 mulheres com a forma clássica da HAC 21OH provenientes de 4 centros médicos. Dados clínicos, hormonais e o grau de virilização genital foram obtidos de forma retrospectiva da análise de prontuários. A intensidade de virilização genital foi classificada de acordo com a escala de Prader (P) e as pacientes foram divididas em 4 grupos: P I+II, P III, P IV e P V. As pacientes também foram agrupadas de acordo com o genótipo 21OH: grupo A carreadoras de mutações que predizem < 2% de atividade enzimática residual (n= 122) e grupo B carreadoras de mutações que predizem 3 a 7% de atividade residual (n= 58). Metodologias: foram amplificadas e re-sequenciadas as regiões que flanqueiam os exons dos genes CYP3A7, PXR e CAR. Para as variantes funcionais, foram re-sequenciados os exons 12-13 do POR e a região promotora do HSD17B5. As variantes V89L e A49T do SRD5A2 foram rastreadas por PCR-RFLP e o nCAG do RA por eletroforese capilar e análise pelo GeneScan. A determinação da expressão gênica em pele genital foi feita por PCR em tempo real utilizando os genes endógenos CYC, PGK1 e B2M. Os testes t-test, Mann-Whitney, Kruskal-Wallis, Fisher e regressão linear uni- e múltipla foram utilizados na análise estatística. Resultados: o Prader score no genótipo A variou de II a V (III: III IV) e no genótipo B de I a V (III: II - III) (P< 0,001). Foram encontradas em 2,5% dos alelos a variante CYP3A7*1C, em 24% CYP3A7*2, 31% rs2307424 CAR, 25% A503V POR, 33% -71G HSD17B5, 17% rs2518047 HSD17B5, 31% V89L SRD5A2 e em 1% dos alelos a variante A49T SRD5A2. Foi identificada associação das variantes rs2307424 CAR (P= 0,023 ;r2= 0,253) e rs2518047 HSD17B5 (P= 0,006; r2= 0,144) com o grau de virilização genital, tem sido encontradas em maior frequência no grupo de pacientes com virilização mais intensa. Todas as outras variantes não apresentaram associação com o Prader score (P> 0,05). As diferenças de expressão de todos os genes analisados em amostras de pele genital não foram estatisticamente significantes (P> 0,05), embora observou-se que em 4/7 amostras de pacientes com Prader score IV houve uma super-expressão do gene SRD5A2 em relação a 1/5 pacientes com Prader score III. Conclusão: neste estudo multicêntrico observamos que o genótipo 21OH se correlacionou com a intensidade de virilização genital em mulheres com a forma clássica. A variante rs2307424 do gene CAR, relacionada ao metabolismo pré-natal de andrógenos, e a variante rs2518047 do gene HSD17B5, relacionada à síntese de testosterona, associaram-se à fenótipos de virilização mais intensos. Não identificamos diferenças na expressão tecidual dos genes relacionados à síntese e/ou ação periférica de andrógenos em pacientes com os diferentes graus de virilização / Congenital Adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency (21OH) is an autosomic recessive disorder characterized by an impairment in the cortisol and/or aldosterone synthesis, being the most frequent cause of 46,XX disorder of sex development. The disease presents a wide phenotypic variability resulting from different CYP21A2 gene mutations and a strong correlation has been observed among genotypes, clinical forms and basal hormone levels. However, this correlation is not observed regarding the degree of prenatal external genitalia virilization in females and an interindividual variability in the synthesis, metabolism and/or peripheral action of androgens could corroborate for these findings. Objectives: to evaluate if allelic variants in genes related to the androgen synthesis, metabolism and peripheral action could modulate the genital phenotype in CAH females bearing similar CYP21A2 mutations. Differences in the HSD17B5, SRD5A1, SRD5A2 and RA gene expression in genital skin were evaluated among patients with different degrees of external genital virilization. Patients: were selected 187 CAH females and clinical and hormonal data were retrospectively evaluated. The degree of external genitalia virilization was classified according to Prader (P) scores and patients were divided into 4 groups: P I+II, P III, P IV and P V. Patients were also grouped according to 21OH genotypes: group A bearing mutations predicting < 2% of residual enzymatic activity (n= 122) and group B between 3 to 7% (n= 58). Methodology: the exonic flanking regions of CYP3A7, PXR e CAR genes were PCR amplified and sequenced. The exons 12-13 of POR and the promotor region of HSD17B5 were sequenced to screen the functional polymorphisms. The V89L and A49T SRD5A2 alleles were screened by PCR-RFLP and the CAG polymorphic tract of AR gene by capillary electrophoresis and GeneScan analysis. The differential gene expression in genital skin was evaluated by real time PCR and the CYC, PGK1 e B2M housekeeping genes were used. The t-test, Mann-Whitney, Kruskal-Wallis, Fisher and uni- and multiple linear regression tests were used in statistical analysis. Results: Prader score in group A varied from II to V (III: III - IV) and in group B from I to V (III: II - III) (P< 0,001). The CYP3A7*1C allele was identified in 2.5% of alleles, CYP3A7*2 in 24%, rs2307424 CAR in 31%, A503V POR in 25%, -71G HSD17B5 in 33%, rs2518047 HSD17B5 in 17%, V89L SRD5A2 em 31% and A49T SRD5A2 in 1% of alleles. The rs2307424 CAR (P= 0.023; r2= 0.253) and rs2518047 HSD17B5 variants (P= 0.006; r2= 0.144) were associated with the degree of external genitalia virilization, and they were found in a higher frequency in more virilized patients. The remaining variants were not associated with Prader scores (P> 0.05). The HSD17B5, SRD5A1, SRD5A2 and RA gene expressions did not significantly differ between patients presenting Prader score III and IV (P> 0.05); however, 4/7 samples from patients with Prader IV and just 1/5 patients with Prader III presented an increased SRD5A2 expression. Conclusion: In this multicentric study the 21OH genotypes were correlated with the degree of external genitalia virilization in CAH females. The rs2307424 CAR and the rs2518047 HSD17B5 variants, related to the prenatal androgen metabolism and synthesis, respectively, explained some of the interindividual variability of genital phenotype in CAH females bearing similar CYP21A2 mutations. Differences in the expression of genes involved in the peripheral androgen action did not corroborate for the variability of genital phenotype in CAH

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No description available.

<p>Disease-specific compounds (biomarkers) are analyzed in clinical laboratories to assist with diagnosing diseases. This thesis describes development and validation of liquid chromatography tandem mass spectrometry (LC-MS/MS) based tests for diagnosing a diverse group of endocrine and metabolic diseases. The analytical methods used on-line and off-line sample extraction and analytical derivatization as means of enhancing the analytical sensitivity, specificity and clinical utility. All developed methods were extensively validated and reference intervals for the biomarker concentrations were established in blood samples of healthy adults and children. Advantages of the LC-MS/MS as an analytical technique include possibility of simultaneous measurement of multiple analytes and ability of confirming their identity. In this thesis we proposed and evaluated approaches for the assessment of the specificity of analysis in the methods that use tandem mass spectrometry detection. To enhance throughput of the LC-MS/MS tests for the biomarkers that have endogenous or exogenous isomers an approach was developed for quantitation of isomers from unresolved chromatographic peaks. Using methods developed in this thesis we performed a study of the steroidogenesis in ovarian follicles of healthy women and women with polycystic ovary syndrome (PCOS). Obtained data on the steroid concentrations and associations between the steroid metabolites in the pathway would be helpful for better understanding of the ovarian pathophysiology. Potential biomarkers of PCOS were identified in the thesis; further studies will be necessary to confirm their clinical utility.</p>

L’hyperplasie bilatérale macronodulaire des surrénales (HBMS) conduit au développement de nodules corticosurrénaliens bilatéraux entraînant un syndrome de Cushing diagnostiqué souvent au cours de la cinquième décennie. L’étiologie de cette maladie n’était que partiellement connue mais le caractère bilatéral de l’atteinte surrénalienne et l’observation de cas familiaux suggéraient, au début de ce travail, une origine génétique. L’analyse de l’ADN tumoral et leucocytaire d’une série de 33 patients opérés a permis de mettre en évidence chez 25% des patients une perte d’hétérozygotie neutre en nombres de copies (LOH) de tout le bras court du chromosome 16 (16p) au niveau du tissu surrénalien. Un séquençage complet du génome de 5 patients (ADN germinal et tumoral) a permis de mettre en évidence une mutation du gène ARMC5 localisé en 16p pour 4 patients. Le séquençage direct des parties codantes d’ARMC5 à partir de l’ADN somatique et germinal de l’ensemble des patients opérés de la cohorte a montré qu’au total, 55% des patients avaient une mutation d’ARMC5. L’inactivation du gène se fait selon la théorie de Knudson (un événement germinal inactivant le premier allèle associé à un autre événement somatique inactivant le second allèle) ce qui laisse supposer qu’ARMC5 est un gène suppresseur de tumeur. L’analyse de la cohorte d’HBMS de nos collaborateurs américains au National Institute Health (laboratoire de CA. Stratakis, Bethesda, USA) a permis de confirmer que les mutations d’ARMC5 étaient un événement fréquent. Des variants de ce gène sont aussi associés à l’hypertension à rénine basse chez les patients noirs-américains. Afin de déterminer des corrélations génotype-phénotype, notre cohorte initiale a été élargie pour constituer une série consécutive de 98 cas index de patients présentant des formes légères à sévères de la maladie, opérées ou non. Vingt-quatre patients (25%) présentaient une altération d’ARMC5. Par ailleurs, 31 nodules surrénaliens de 19 patients ont été analysés en somatique. Le second événement était une mutation dans 68% des cas et une LOH du locus pour les 32% restant. Chez un même patient, le second événement était différent dans chaque nodule présenté. Les patients mutés avaient un syndrome de Cushing plus sévère cliniquement et biologiquement par rapport aux patients non mutés. La taille de leurs surrénales étaient plus grandes avec un plus grand nombre de nodules. Les patients mutés étaient aussi plus jeunes au diagnostic et plus souvent hypertendus. Ces patients étaient ainsi plus souvent opérés. La fonction de la protéine ARMC5 n’était pas connue lors de son identification comme gène de l’HBMS. In vitro, la surexpression du gène sauvage induit l’apoptose. La surexpression des mutants faux-sens et du mutant p.F700del retrouvés chez les patients entraîne moins d’apoptose qu’ARMC5 sauvage. La protéine ARMC5 contient des domaines Armadillo et BTB, connus pour être impliqués dans l’interaction protéine-protéine. En physiologie, l’ACTH stimule la production d’AMPc et la voie de la protéine kinase A (PKA) est impliquée dans différentes pathologies corticosurrénaliennes. Nous avons pu montrer qu’ARMC5 interagissait avec la sous-unité catalytique alpha de la PKA. L’invalidation d’ARMC5 conduit in vitro à une diminution de l’expression d’enzymes de la stéroïdogénèse, de la production de cortisol et une diminution de l’activité PKA. Ainsi, l’hypothèse pour expliquer les HBMS liées à une inactivation d’ARMC5 est que la perte d’apoptose conduit à une hyperplasie nodulaire du tissu corticosurrénalien et que, même si la production de cortisol est diminuée à l’échelle unicellulaire, l’effet de masse global conduit au total à un hypercortisolisme. Nos travaux ont donc identifié et caractérisé un premier gène causal, ARMC5, fréquemment impliqué dans l’HBMS et associé à des formes plus sévères de la maladie. Cette découverte ouvre des perspectives pour le diagnostic familial et la prise en charge des patients. (...) / Primary bilateral macronodular adrenal hyperplasia (PBMAH) is a rare cause of adrenal Cushing’s syndrome and bilateral adrenal tumors. We suspected a genetic origin of the disease on the basis of the report of some familial cases and the involvement of both adrenal glands. The aim of this study was to find a genetic cause of non syndromic PBMAH. To look at chromosomal abnormalities, we use single-nucleotide polymorphism (SNP) arrays and microsatellite markers analysis in a first series of 33 patients all operated for PBMAH. We realize whole genome sequencing of 5 patients (blood and tumor DNAs matched). Then we genotyped by Sanger sequencing the gene Armadillo Repeat Containing 5 (ARMC5) in this first series and 66 additional patients. Clinical data were collected to establish genotype-phenotype correlation. In addition, the cohort of patients of our collaborators at the National Institute Health (Dr. Stratakis, Bethesda, USA) was studied. The effects of ARMC5 inactivation and overexpression and the partners of the protein were sought in cell-culture models. The most frequent somatic alteration was a loss of heterozygosity at 16p observed in tumors of 25% of the patients. The gene ARMC5, located at 16p11.2, was the most frequently mutated by whole genome sequencing: a mutation was found in 4/5 patients. 55% of the patients of the first cohort (33 patients treated by adrenalectomy for PBMAH) had ARMC5 alteration. One patient presented with germline microdeletion of the locus identified by SNP array. Every patient had two events: either a mutation or a deletion at the germline level, either a second mutation or a LOH at the somatic level. We showed that the two events were present on different alleles suggesting that ARMC5 is a tumor suppressor gene. In addition, we showed for several patients that the second hit was different in each adrenal nodules of a same patient. This first cohort included only operated patients with serious forms of the disease. The study of the American cohort and the analysis of the total cohort of our lab including non-operated patients and milder forms showed an alteration of ARMC5 in about 25% of the patients. Genotype-phenotype correlation showed that ARMC5 defects are associated with younger age at the diagnosis, higher hypercortisolism, bigger adrenals and higher number of nodules. In addition, a mutation of ARMC5 was shown in a patient with a PBMAH secreting both aldosterone and cortisol. Analysis of a series of patient affected by primary hyperaldosteronism suggested that ARMC5 may be associated with hypertension especially in African-American subjet. Overexpression of ARMC5 leads in vitro to cell apoptosis. We showed that this apoptosis was reduced when transfecting vector harboring missense mutations or single amino-acid deletion found in our cohort. Invalidation of ARMC5 leads to a decreased steroidogenic enzymes expression, cortisol production and reduced protein kinase A (PKA) activity. We showed that ARMC5 interacts with the calaytic subunit alpha of the PKA dissociated from the cAMP-bound regulatory subunits. More than one quarter of sporadic PBMAH patients present a pathogenic germline ARMC5 defect and these index cases present a more severe disease. Systematic genotyping of ARMC5 may help for early diagnosis of PBMAH, familial counseling, and patients’ management. ARMC5 appears to be a new regulator of PKA and might represent a new target for the development of pharmacological agents controlling PKA function and cortisol production.

A hiperplasia adrenal congênita (HAC) é um grupo de doenças hereditárias causadas por uma deficiência em uma das enzimas necessárias para a síntese de cortisol no cortex adrenal. Mais de 95% de todos os casos de HAC são devidos a 21-Hidroxilase (21-OHD). Existem 3 formas principais, duas com manifestações clínicas no período neonatal, a forma mais grave perdedora de sal (HAC-PS) e a forma virilizante simples (HAC-VS). Além da perda salina, o excesso de andrógenos leva à virilização de recém nascidas femininas. As formas neonatais são chamadas de clássicas, atividade enzimática da 21-OH bastante reduzida, de < 2% na HAC-PS e de 2-10% na HAC-VS. A forma parcial de início tardio é chamada de HAC não clássica (HAC-NC) e a principal manifestação na infância é a adrenarca precoce. Nesta forma a atividade da 21-OH é de 20-60%. Os programas de triagem para HAC visam, principalmente, ao diagnóstico precoce da forma clássica perdedora de sal, mais grave e potencialmente letal. No Brasil, a triagem pública é realizada no Estado de Goiás desde 1997 e em Santa Catarina desde 2001. No Rio Grande do Sul (RS) foi implantada em maio de 2014 na fase IV do Programa Nacional de Triagem Neonatal. A inclusão da HAC trouxe consigo muitos desafios e a exigência de um fluxo de triagem e diagnóstico bem estruturados. O diagnóstico precoce é crucial para prevenir o óbito de lactentes por insuficiência adrenal. Dosa-se, em papelfiltro, a 17OH progesterona (17-OHP). Elevações podem ocorrer em recém-nascidos sem HAC (falso-positivos), devido a situações de estresse perinatal e por prematuridade. Após avaliação dos dados do primeiro ano de triagem para HAC neste estudo, a mediana da idade da coleta nos casos diagnosticados foi de 8 dias (4.25-15.75). Dos 8 casos diagnosticados de maio de 2014 a abril de 2015, 6 casos com forma perdedora de sal (incluindo 1 caso de óbito por coleta tardia do TP aos 38 dias de vida). A incidência encontrada em nosso meio no primeiro ano foi de 1:13.551 casos. Com a estratégia do uso de pontos de corte estratificados pelo peso de nascimento18, o índice total de resultados positivos em nosso meio foi de 0,5% da amostra avaliada (“n” total de 514 bebês), sendo mais frequente em recém nascidos com menos de 2000g de peso ao nascer. Além da confirmação clínica e laboratorial, o genótipo é importante, além de confirmar, para estabelecer gravidade da doença e também para ratificar o diagnostico dos falsos positivos na ausência de uma mutação do gene CYP21A2. Um dos casos confirmados de HAC-PS foi associado a múltiplas malformações e craniossinostose severa, suscitando a hipótese de um a associação com defeito de FGFR2. A correlação genótipo- fenótipo na avaliação dos casos em dois anos da triagem alcançou um alto nível de concordância de 87%. Diagnosticada, portanto, de forma assertiva a HAC forma clássica, instituise a terapia glicocorticóide para as formas virilizante simples e acrescenta-se mineralocorticóide para as formas perdedoras de sal. A triagem neonatal é um importante programa de saúde populacional e visa ao diagnóstico precoce de uma patologia com potencial risco à vida pela perda de sal, além de permitir adequada atribuição de sexo nas meninas com virilização genital e à saúde da criança. Estabelecer os fluxos adequados de triagem e manejo, além de ampliar o conhecimento sobre a HAC, com o reconhecimento dos desfechos e tratamentos adequados é essencial para minimizar as possíveis complicações nesta população de maior vulnerabilidade. / Congenital adrenal hyperplasia (CAH) is a group of inherited diseases caused by a deficiency in one of the enzymes required for the cortisol synthesis by the adrenal cortex. More than 95% of all CAH cases are due to 21-hydroxylase (21-OHD). There are 3 forms, two with neonatal clinical manifestation: salt-wasting CAH (SW-CAH\) and the simple virilizing form (HAC-VS). In addition to salt loss, androgens excess lead to the virilization of female newborn. Neonatal forms are defined as classical CAH. The 21-OHD enzymatic activity in SWCAH is less than <2% and in the SV-CAH 2-10%. A late-onset form, with partial enzymatic defect (20-60%) is called non-classical HAC (NC-CAH) and the main manifestation in childhood is early adrenarche. In Brazil, public health screening has been conducted in the State of Goiás since 1997 and in te Sate of Santa Catarina since 2001. In Rio Grande do Sul (RS) it was implemented in May 2014, in phase IV of the National Neonatal Screening Program. The inclusion of CAH in the local screening program brought many challenges and the need of a well structured screening and diagnosis flowchart. Early diagnosis is crucial to prevent infant death due to adrenal insufficiency. Around de world, the screening programs for CAH main purpose is the early diagnosis of the more severe classical forms, especially SW-CAH. The cortisol precursor 17OH progesterone (17-OHP) is the main disease marker and is measured on filter paper. Elevations may occur in infants without CAH (false positive) due to perinatal stress and prematurity. Of newborns screened in the first year, median age of collection in diagnosed cases was 8 days (4.25-15.75) and 8 patients were diagnosed with CAH (four males, four females). The incidence of CAH in the state was 1:13,551. Six cases were identified as classic salt-was-ting CAH and two were cases of virilizing CAH. The overall rate of positive results was 0.5% (n = 514 infants). The number of false positive results was higher among newborns with birth weight < 2,000 g. In addition to clinical and laboratory confirmation, the genotype is important to confirme 21-OH deficiency, to establish disease severity and also in the absence of a mutation of the CYP21A2 gene to more precise exclude the diagnosis of suspected false positives. One of the confirmed cases of SW-CAH was associated with multiple malformations and severe craniosynostosis, raising the hypothesis of an association with FGFR2 mutation. A high genotype- phenotype correlation of 87% was found in the cases after two years of screening. Once the classic CAH is diagnosed, glucocorticoid therapy is instituted and mineralocorticoid is added for SW-CAH. CAH neonatal screening is an important population health program and aims to the early diagnosis of a pathology with a potential risk due to salt loss crisis. The early detection of cases also allows to adequate sex assignment in girls with genital virilization. Establishing adequate screening flows, proper diagnosis and management, in addition to increase knowledge about the disease, with the appropriate recognition of outcomes and treatments is essential to minimize complications in this population of greater vulnerability.