21-hydroxylase deficiency is the commonest enzyme defect in congenital adrenal hyperplasia resulting in low circulating cortisol and, in severe cases, low aldosterone which may lead to cardiac arrest in the neonate. As the low levels of coitisol· lead to raised levels of adrenocorticotrophic hormone large concentrations of androgens are formed in utero which may lead to cliteromegaly and consequent confusion of the sexes at birth. High concentrations of the androgens give rise to rapid growth in early childhood and early fusion of the epiphyses results in short stature of the adult. 17-alphahydroxyprogesterone (170HP) is raised in the blood and saliva of children with 21-hydroxylase deficiency. 170HP is commonly measured by radioimmunoassay which limits the assay to being performed by laboratories equipped with beta or gamma counters. The aim of this project was to develop an enzymeimmunoassay which could be carried out using the minimum amount of equipment. Horseradish peroxidase was conjugated to 170HP-O-carboxymethyloxime using a mixed anhydride reaction. Separation of free and bound label was achieved using a second antibody linked to magnetisable particle solid phase. Measurement of the bound enzyme activity yielded colours which could be compared by eye, with known standards, to give a semi-quantitative assay. Alternatively the absorbance could be measured using a spectrophotometer for full quantitative results. The developed assays were evaluated, comparing the results with radioimmunoassay, and then used for measuring 170HP in blood and saliva in various clinical situations.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst., 2001. / Härtill 5 uppsatser.

We present a case of a woman with a history of Congenital Adrenal Hyperplasia (CAH) diagnosed at the age of 12, who was referred to our unit for surgical treatment. Despite the initial diagnosis was an indirect inguinal hernia, it was a misdiagnosis. Once in our service, this was corrected into clitoromegaly secondary to CAH. Physical examination and imaging test discarded other abnormalities, such as secondary effects androgenization. Regarding surgical treatment, the techniques used were Spencer and Allen combined with Kumar, which are the most used for clitoroplasty but also less used in Peru.

Thesis advisor: Charles S. Hoffman / The biological roles of the most recently discovered mammalian cyclic nucleotide phosphodiesterase (PDE) family, PDE11, are poorly understood, in part due to the lack of selective inhibitors. To address this need for such compounds I completed a ~200,000 compound high throughput screen (HTS) for PDE11 inhibitors using a yeast-based growth assay. Further characterization of lead candidates using both growth-based assays in the fission yeast Schizosaccharomyces pombe and in vitro enzyme assays identified four potent and selective PDE11 inhibitors. I examined the effect of these compounds on human adrenocortical cells, where PDE11 is believed to regulate cortisol levels. One compound, along with two structural analogs, elevates cAMP levels and cortisol production through PDE11 inhibition, thus phenocopying the behavior of adrenocortical tumors associated with Cushing syndrome. These compounds can be used as research tools to study the biological function of PDE11, and can also serve as leads to develop therapeutic compounds for the treatment of adrenal insufficiencies. This study further validates the yeast-based HTS platform as a powerful tool for the discovery of potent, selective and biologically-active PDE inhibitors. / Thesis (PhD) — Boston College, 2012. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Diss. (sammanfattning) Stockholm : Karol. inst., 2005. / Härtill 5 uppsatser.

Thousands of animal experiments have demonstrated that androgenic hormones, such as testosterone, during the prenatal and early postnatal periods, masculinise and defeminise various neural and behavioural characteristics that differ by sex. Can these findings from animal experiments be generalised to human behaviour? Can early androgen exposure shape subsequent gender-related disorders in humans? Chapter 1 (Introduction) provides an overview of the literature. Chapter 2 (Kung et al., 2016a) is the first study to demonstrate that testosterone concentrations in saliva samples collected during the early postnatal testosterone surge at 1 to 3 months of age can negatively predict subsequent expressive vocabulary size (how many words a child can say) during toddlerhood. Notably, males typically have a smaller expressive vocabulary than do females during toddlerhood and a small expressive vocabulary is predictive of subsequent language difficulties, such as dyslexia and stuttering, which are more common in boys. Chapters 3 (Kung et al., 2016b) and 4 (Kung et al., 2016c) evaluate a popular theory of autism, the extreme male brain theory, which argues that heighted androgen exposure during early development causes the male preponderance in autism. To test the hypothesised relationship, Chapters 3 and 4 use different measures and study populations, including testosterone concentrations in amniotic fluid samples obtained prenatally and saliva samples obtained during the early postnatal testosterone surge in typically developing children, as well as examining the adjustment in children exposed to unusually high levels of androgens prenatally due to congenital adrenal hyperplasia (CAH), a rare clinical condition occurring in approximately 1 in 18,000 births. Findings from these two chapters converge to show that any relationship between early androgen exposure and subsequent development of autistic traits is small, non-existent, or unreliable, providing a much-needed clarification of the role of early androgen exposure in the aetiology of autism. Using data from a general population study, Chapter 5 (Kung et al., 2018a) is the first study to show that male-typical play behaviour in early childhood, a trait that has been linked to increased early androgen exposure in previous research, can positively predict adolescent physical aggression, which is typically higher in males than in females. This positive association between play and aggression supports potential influences of early androgen exposure, as well as socio-cognitive influences involved in gender development. Chapter 6 (Kung et al., 2018b) is the first study to compare emotional and behavioural adjustment in children with CAH, their unaffected siblings, and children in the general population. Findings from this chapter suggest that although within the families with a child with CAH there are generally no differences in emotional or behavioural problems between boys or girls with CAH and their unaffected same-sex siblings, both girls with CAH and their unaffected sisters are at risk of developing behavioural problems when compared with girls in the general population. Familial influences and social stigma may contribute to this gender-specific pattern of behavioural adjustment. Finally, Chapter 7 (Discussion) integrates the findings and previous research and provides directions for further research. Chapter References Chapter 2 Kung, K. T. F., Browne, W. V., Constantinescu, M., Noorderhaven, R. M., and Hines, M. (2016). Early Postnatal Testosterone Predicts Sex-Related Differences in Early Expressive Vocabulary. Psychoneuroendocrinology, 68, 111-116. Chapter 3 Kung, K. T. F., Constantinescu, M., Browne W. V., Noorderhaven, R. M., and Hines, M. (2016). No Relationship Between Early Postnatal Testosterone and Autistic Traits in 18 to 30-Month-Old Children. Molecular Autism, 7:15. Chapter 4 Kung, K. T. F., Spencer, D., Pasterski, V., Neufeld, S., Glover, V., O'Connor, T. G., Hindmarsh, P. C., Hughes, I. A., Acerini, C. L., and Hines, M. (2016). No Relationship Between Prenatal Androgen Exposure and Autistic Traits: Convergent Evidence from Studies of Children with Congenital Adrenal Hyperplasia and of Amniotic Testosterone Concentrations in Typically-Developing Children. Journal of Child Psychology and Psychiatry, 57, 1455-1462. Chapter 5 Kung, K. T. F., Li, G., Golding, J., and Hines, M. (2018). Preschool Gender-Typed Play Behavior at Age 3.5 Years Predicts Physical Aggression at Age 13 Years. Archives of Sexual Behavior, 47, 905-914. Chapter 6 Kung, K. T. F., Spencer, D., Pasterski, V., Hindmarsh, P. C., Neufeld, S. A. S., Hughes, I. A., Acerini, C. L., and Hines, M. (2018). Emotional and Behavioral Adjustment in 4- to 11-Year-Old Boys and Girls with Classic Congenital Adrenal Hyperplasia and Unaffected Siblings. Psychoneuroendocrinology. 97, 104-110.

Orientador: Gil Guerra Junior / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-10T14:57:15Z (GMT). No. of bitstreams: 1 Goncalves_EzequielMoreira_M.pdf: 3646841 bytes, checksum: 92edeb1af3250e23a5dd5ef4630bd584 (MD5) Previous issue date: 2008 / Resumo: A Hiperplasia Adrenal Congênita na forma clássica da deficiência da 21-hidroxilase (HAC-C-D21OH) caracteriza-se pela deficiência na síntese do cortisol e, em alguns casos, na aldosterona, e pelo excesso de andrógenos. O objetivo do estudo foi avaliar a composição e as proporções corporais de pacientes do sexo feminino com HAC-C-D21OH e comparar em relação ao controle, à forma clínica da doença e a um grupo controle de ambos os sexos. A casuística foi composta por 28 pacientes (grupo casos) do sexo feminino com HAC-C-D21OH divididas de acordo com o controle hormonal (bem e mal controladas) e com a forma da doença (virilizante simples e perdedores de sal). O grupo controle foi composto por 112 indivíduos saudáveis de ambos os sexos, divididos em dois grupos, de acordo com o gênero. Para verificar os efeitos da puberdade nas medidas avaliadas, os três grupos foram subdivididos em três faixas etárias: até 10 anos, de 11 a 14 anos e acima de 15 anos. Para a avaliação das proporções e composição corporais foram utilizadas medidas antropométricas e a impedância bioelétrica. Os dados das medidas antropométricas foram convertidos em escores de desvio padrão (z). Em relação à forma clínica da doença, as virilizantes simples apresentaram valores significativamente maiores de z do IMC, do perímetro da cintura e da área gorda do braço, e no percentual de massa gorda. Em relação ao controle laboratorial, as mal controladas apresentaram z do comprimento da perna significativamente menor. Os casos apresentaram valores significativamente maiores que os controles no z do IMC, área muscular e gorda do braço, perímetro braquial, da cintura e do quadril, do diâmetro biilíaco e no percentual de massa gorda, e menores no z do comprimento da perna e da mão e nos percentuais de água e massa magra. Pode-se notar que as alterações na composição corporal foram observadas em todas as faixas etárias, mas foram se intensificando com a idade, com o predomínio do aumento da massa gorda enquanto as alterações nas proporções corporais se tornaram mais evidentes após a puberdade, com comprometimento principal das extremidades (perna, mão e pé). As variáveis que melhor se ajustaram para diferenciar os grupos caso e controles foram o percentual de gordura, o comprimento da mão e o perímetro do quadril (94,7%). Esse grupo de pacientes apresentou diferenças nas proporções e principalmente na composição corporal, justificando uma avaliação antropométrica ampla e cuidadosa no monitoramento das pacientes com HAC-C-D21OH / Abstract: Congenital Adrenal Hyperplasia due to classical 21-hydroxylase deficiency (CAH-C-21OHD) is characterized by deficiency in the synthesis of cortisol, and, in some cases, also of aldosterone and by excess of androgens. The aim at of the study was to evaluate the body composition and body proportion of women with CAH-C-21OHD in relation to the control and the clinical form of the disease and to a group control of both sexes. The casuistic was composed by twenty-eight patient (cases group) female with CAH-C-21OHD were divided according to control (good or bad) and the clinical form of the disease (salt-wasting and simple-virilizing). The control group was composed by 112 healthy individuals of both sexes, divided in 2 groups, in agreement with the gender. To verify the effects of the puberty in the appraised measures, the 3 groups were subdivided in 3 age groups: up to 10 years, from 11 to 14 years and above 15 years. To evaluate body proportions and composition, anthropometric measurements and electric impedance were analyzed. The data of anthropometric measurements were converted in standard deviation scores (z). In relation to clinical form of the disease, the simple-virilizing presented values significantly larger of z of BMI, of the perimeter of the waist and of the fat area of the arm, and in the percentage of fat mass. In relation to disease control, the patients with bad control showed significantly lower leg length. The cases showed significantly higher values in relation to controls in z of BMI, arm muscle and fat area, brachial perimeter, of the waist and of the hip, of the biiliac diameter and in the fat percentage and smaller in z of the length of the leg and of the hand and in the percentages of water and lean mass. Alterations in the body composition were observed in all of the age groups, but they were if intensifying with the age, with the prevalence of the increase of the fat mass while the alterations in the body proportions if they turned more evident after the puberty, with main compromising of the extremities (leg, hand and foot). The more adjusted variables to explain the differences between cases and controls were percentage of fat mass, hand length and hip perimeter, comprising 94.7% of the differences between the groups. This group of patients showed differences in body proportions, but mainly in body composition, justifying a broad and careful anthropometric evaluation in the monitoring of CAH-C-21OHD patients / Mestrado / Saude da Criança e do Adolescente / Mestre em Saude da Criança e do Adolescente

Orientadores: Maricilda Palandi de Mello, Anna Wedell / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T13:24:54Z (GMT). No. of bitstreams: 1 Soardi_FernandaCaroline_D.pdf: 4267789 bytes, checksum: 80c77e1664dcc041014d42a92bdd435e (MD5) Previous issue date: 2008 / Resumo: A causa mais freqüente de hiperplasia congênita da adrenal (HCA) é a deficiência da enzima CYP21A2 responsável por cerca de 90% dos casos, seguida da deficiência de CYP11B1, a qual é responsável por 5-8%. A deficiência de CYP21A2 apresenta diferentes sintomas clínicos, que podem variar de uma forma leve não clássica (NC) a uma forma grave clássica, dividida em virilizante simples (VS) e perdedora de sal (PS). Enquanto a deficiência de CYP11B1 é classificada nas formas clássica e não-clássica, dependendo da gravidade do fenótipo. As diferentes formas destas deficiências estão associadas a mutações distintas ou a combinação de mutações nos genes, sendo estas mutações provenientes dos genes homólogos ou não. O primeiro objetivo desta tese foi identificar novas mutações em alelos de 31 pacientes. As variantes protéicas novas p.G56R, p.L107R e p.L142P e, as raras p.H62L, p.H62L+p.P453S e p.R408C do gene CYP21A2 foram expressas para comparar as atividades da enzima CYP21A2 nas suas formas normal e mutantes. Foi objetivo, também, estudar através da técnica de mini-genes as possíveis alterações no processo de splicing para as variantes IVS2+5G>A, IVS2-2A>G, IVS4- 15A>C+IVS4-8C>T+p.D183E do gene CYP21A2 e para a variante g.1753G>A do gene CYP11B1. O estudo da atividade enzimática do gene CYP21A2, realizado pela técnica de mutagênese sítio-dirigida, demonstrou que as mutações p.L107R, p.L142P e p.R408C reduziram a atividade enzimática para valores praticamente nulos, classificando-as como responsáveis pela forma PS. A alteração p.G56R apresentou uma quantidade mínima de conversão de progesterona em desoxicorticosterona, quantidade suficiente para evitar a perda de sal, sendo considerada clássica associada à forma VS. A mutação p.H62L foi encontrada no mesmo alelo que a mutação p.P34L, em uma das pacientes da casuística desse trabalho, ambas inseridas num gene quimérico portador da deleção de 30 Kb. A mutação p.H62L também foi encontrada em associação com a mutação p.P453S, em dois pacientes de origem Escandinava. No estudo funcional a mutação p.H62L reduziu parcialmente a atividade enzimática. Os resultados cinéticos classificaram essa mutação como relacionada à forma NC da deficiência de CYP21A2. Em combinação com a mutação p.P453S, observou-se um sinergismo, uma vez que reduziu a atividade da enzima para a faixa limítrofe entre NC e VS. A investigação no processo de splicing utilizando a técnica de minigenes para as alterações no gene CYP21A2 indicou que a variação IVS2+5G>A causa a perda do exon 2 na formação do mRNA, sendo relacionada à forma PS. Da mesma forma, a variação IVS2- 2A>G foi classificada como associada à forma PS, pois inseriu no mRNA 19 bases provenientes do intron 2 na junção exon2-exon3, o que modificou o frame de leitura do mRNA criando um códon de parada prematura. Por outro lado, ficou demonstrado que as variações IVS4- 15A>C+IVS4-8C>T+p.D183E não interferem no processamento normal do mRNA do gene CYP21A2. No caso da alteração g.1753G>A no gene CYP11B1, que foi classificada como responsável pela forma clássica da deficiência de CYP11B1, o estudo de mini-gene indicou a perda dos últimos 45 nucleotídeos do exon 4, criando um códon de parada prematura. A elucidação do papel funcional e estrutural das mutações nos genes estudados permitiu o correto estabelecimento da correlação genótipo-fenótipo na maioria dos pacientes com HCA estudados / Abstract: Deficiency of CYP21A2 enzyme is responsible for more than 90% of congenital adrenal hyperplasia (CAH) followed by the deficiency of CYP11B1, which is responsible for 5-8% of the cases. The deficiency of CYP21A2 is normally classified in clinical forms that vary from a mild non-classical (NC) to a severe classical form, which can manifest as salt wasting (SW) or as simple virilizing (SV). Depending on the severity of phenotype, deficiency of CYP11B1 can be classified in classical or non-classical forms. In both deficiencies the clinical forms are associated with different mutations or combination of mutations, which may or may not be originated from the homologous genes. The aim of this study was to identify novel or rare mutations in alleles of 31 patients with CYP21A2 deficiency. Using site-direct mutagenesis strategies, nucleotide variants were introduced into the cDNA and the novel p.G56R, p.L107R and p.L142P and rare p.H62L, p.H62L+p.P453S and p.R408C protein variants of CYP21A2 were expressed to compare the enzymatic activity between the wild-type and mutant proteins. Furthermore, splicing activities were investigated for IVS2+5G>A, IVS2-2A>G, IVS4-15A>C+IVS4-8C>T+p.D183E sequence CTP21A2 variations and for g.1753G>A on CYP11B1 gene by minigene constructions. The analysis of enzymatic conversion of both CYP21A2 substrates, 17-hydroxyprogesterone and progesterone, into 11-desoxycortisol and corticosterone, respectively, showed low levels of residual activities for p.L107R, p.L142P and p.R408C, which were classified as SW mutations. Whereas, the result of enzyme activity for p.G56R indicated that it might be a SV-related mutation due a residual activity of 1.4% toward progesterone as substrate. The p.H62L was associated to p.P34L mutation in a chimeric gene present in a 30-kb deletion allele in Brazilian patients. In Scandinavian patients, the p.H62L mutation was found associated to the p.P453S which is known as a NC mutation. The p.H62L itself showed an activity within the range of NC mutations. The apparent kinetic constant confirmed this classification. A synergistic effect was observed for the allele bearing the p.H62L+p.P453S combination as it had caused a significant reduction in the enzymatic activity bringing it to the borderline level between SV and NC mutations. On the minigene analyses for CYP21A2, the IVS2+5G>A variation showed skipping of exon 2, therefore this alteration was classified as SW mutation. Likely, IVS2-2A>G was considered as a SW mutation due to the insertion of 19 nucleotides from intron 2 into the resulting mRNA, which changed the reading frame and created a premature stop codon. Conversely, the group of variations IVS4-15A>C+IVS4-8C>T+p.D183E did not affect the normal splicing of CYP21A2 mRNA. In the CYP11B1 minigene analysis, the g.1753G>A nucleotide variation was classified as responsible for the classical form of deficiency. An alternative splicing due to disruption of the normal donor site was used and the skipping of the last 45 nucleotides of exon 4 was observed. This alteration modified the mRNA reading frame and created a premature stop codon. The elucidation of functional and structural characters of the steroidogenic gene mutations led to the establishment of a correct genotype-phenotype in most patients studied. / Doutorado / Genetica Animal e Evolução / Doutor em Genetica e Biologia Molecular

Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is the most common inherited endocrinopathy. This disorder is associated with many complications caused either by illness itself or inadequate treatment. The late consequence of this disorder is a reduced final height. Absences of cortisol, androgen excess, with or without salt wasting, virilization of external genitalia in new-born girls are main findings. Androgen excess causes virilization in girls, accelerated bone maturation and early epiphyseal fusion in both sexes. CAH patients are dependent on lifelong treatment with oral glucocorticoids. Growth retardation and obesity are common side effects resulting from glucocorticoid use. The aim of this thesis was to analyse the success of obtaining a final height that is within a genetic potential regarding to treatment compliance. The other objective was the analysis of auxological profile of CAH patients. This study was mainly focused on the analysis of body height, linear proportionality, the weight parameters, body composition, body fat distribution, skeletal robusticity and body structure. In this study we performed anthropometric examination of 30 CAH patients. 25 of them had completed growth (age: 16,4 to 44,5 years), 12 woman and 11 men and 2 men with karyotype 46XX....