Congenital adrenal hyperplasia: focus on the molecular basis of 21-hydroxylase deficiency

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder caused by defects in one of several steroidogenic enzymes involved in the synthesis of cortisol from cholesterol in the adrenal glands. More than 90% of cases are caused by 21-hydroxylase deficiency, and the severity of the resulting clinical symptoms varies according to the level of 21-hydroxylase activity. 21-Hydroxylase deficiency is usually caused by mutations in the CYP21A2 gene, which is located on the RCCX module, a chromosomal region highly prone to genetic recombination events that can result in a wide variety of complex rearrangements, such as gene duplications, gross deletions and gene conversions of variable extensions. Molecular genotyping of CYP21A2 and the RCCX module has proved useful for a more accurate diagnosis of the disease, and prenatal diagnosis. This article summarises the clinical features of 21-hydroxylase deficiency, explains current understanding of the disease at the molecular level, and highlights recent developments, particularly in diagnosis

A prática em contexto profissional na formação inicial:perspectivas de estudantes:contributos para a configuração profissional do educador de infância

Tese de doutoramento, Educação (Formação de Professores), Universidade de Lisboa, Instituto de Educação, 2014O presente trabalho tem como campo de pesquisa a Formação de Professores, mais especificamente a formação dos Educadores de Infância. Centra-se num olhar atento sobre os contributos da imersão na prática em contexto de trabalho durante a formação inicial, no âmbito da formação profissional do educador de infância. Incrito no paradigma interpretativo, incidindo na perspectiva dos sujeitos, a investigação desenvolveu-se em duas fases, conjugando diferentes metodologias e técnicas de recolha e análise de dados. Numa fase inicial, procedeu-se à realização de entrevistas de grupo a estudantes a frequentar o último ano de uma licenciatura em Educação de Infância e a uma entrevista individual ao supervisor institucional que acompanhou esse grupo de estudantes e respectiva análise de conteúdo. Esta fase passou ainda pela recolha e análise de conteúdo de reflexões semanais escritas e incidentes críticos contidos em portfólios de estudantes da referida licenciatura, onde se incluem os anteriores e a ainda a uma análise documental de legislação específica reguladora da formação e da actividade do educador de infância. Uma segunda fase, consistiu na construção e a aplicação de um questionário a estudantes a frequentar também o último ano de formação inicial em educação de infância em três escolas superiores de educação situadas na região de Lisboa e Vale do Tejo, com o intuito de obter uma visão mais ampla e, em simultâneo, mais focalizada do fenómeno em estudo. Os resultados obtidos quer na primeira, quer na segunda etapa permitiram reunir um conjunto de evidências que apontam para a pertinência específica (e incontornável) da prática em contexto profissional, designadamente, como “espaço e tempo” de tomada de consciência dos significados do que é ser EI, de aquisição de novos saberes e de desenvolvimento de outros, de aprendizagem de modos de agir e de estar essenciais ao exercício da profissão. Foi possível identificar ainda um conjunto de tomadas de consciência indiciadoras de mudanças de crenças e concepções iniciais e, por conseguinte, potenciadoras de mudanças de atitudes profissionais. Por fim, os dados permitiram evidenciar algumas pistas susceptíveis de constituírem suporte para reflexão e eventual reconceptualização da componente de prática em contexto profissional na formação inicial de educadores de infância à luz, designadamente, dos novos modelos de organização curricular emanados do processo de Bolonha.The present work is inserted in the research field of teachers training, in particular the initial training of early childhood educators, focusing on the analysis of the contribution of the practicum. The research was based on the interpretative paradigm, focusing on the perspective of the subjects, and was developed in two stages, combining different methodologies and techniques of data collection and analysis. The first step consisted on group interviews to students of the last year of a graduation in Early Childhood Education and an individual interview to the respective institutional supervisor, followed by the content analysis of these interviews. This stage included also the collection and content analysis of weekly written narratives and critical incidents contained in portfólios of students from this graduation, as well as a documental analysis of the specific legislation about the training and professional activity of early childhood educators. On a second stage, a survey was developed and applied to students also attending the last year of initial training in early childhood education in three institutions of higher education located in the Lisbon region, with the aim of obtaining a wider and more specific perspective of our object of study. The results support the importance of the practicum as a phase of the training process in which the student effectively realizes the meaning of being an early childhood educator, acquires and develops professional knowledge and ways of acting and being. It also allows awarenesses that indicate changes in the initial beliefs and conceptions and that can lead to changes in the professional attitudes. Finally, the data suggest clues that can support a reflection and eventual reconceptualization of the practicum in pre-service, namely in light of the new models of curricular organization of Bolonha.Fundação para a Ciência e a Tecnologia (FCT, Programa PROTEC

Streptococcus pyogenes Causing Skin and Soft Tissue Infections Are Enriched in the Recently Emerged emm89 Clade 3 and Are Not Associated With Abrogation of CovRS.

Although skin and soft tissue infections (SSTI) are the most common focal infections associated with invasive disease caused by Streptococcus pyogenes (Lancefield Group A streptococci - GAS), there is scarce information on the characteristics of isolates recovered from SSTI in temperate-climate regions. In this study, 320 GAS isolated from SSTI in Portugal were characterized by multiple typing methods and tested for antimicrobial susceptibility and SpeB activity. The covRS and ropB genes of isolates with no detectable SpeB activity were sequenced. The antimicrobial susceptibility profile was similar to that of previously characterized isolates from invasive infections (iGAS), presenting a decreasing trend in macrolide resistance. However, the clonal composition of SSTI between 2005 and 2009 was significantly different from that of contemporary iGAS. Overall, iGAS were associated with emm1 and emm3, while SSTI were associated with emm89, the dominant emm type among SSTI (19%). Within emm89, SSTI were only significantly associated with isolates lacking the hasABC locus, suggesting that the recently emerged emm89 clade 3 may have an increased potential to cause SSTI. Reflecting these associations between emm type and disease presentation, there were also differences in the distribution of emm clusters, sequence types, and superantigen gene profiles between SSTI and iGAS. According to the predicted ability of each emm cluster to interact with host proteins, iGAS were associated with the ability to bind fibrinogen and albumin, whereas SSTI isolates were associated with the ability to bind C4BP, IgA, and IgG. SpeB activity was absent in 79 isolates (25%), in line with the proportion previously observed among iGAS. Null covS and ropB alleles (predicted to eliminate protein function) were detected in 10 (3%) and 12 (4%) isolates, corresponding to an underrepresentation of mutations impairing CovRS function in SSTI relative to iGAS. Overall, these results indicate that the isolates responsible for SSTI are genetically distinct from those recovered from normally sterile sites, supporting a role for mutations impairing CovRS activity specifically in invasive infection and suggesting that this role relies on a differential regulation of other virulence factors besides SpeB.info:eu-repo/semantics/publishedVersio

Development of a heptaplex PCR assay for identification of Staphylococcus aureus and CoNS with simultaneous detection of virulence and antibiotic resistance genes

Background Staphylococcal toxicity and antibiotic resistance (STAAR) have been menacing public health. Although vancomycin-resistant Staphylococcus aureus (VRSA) is currently not as widespread as methicillin-resistant S. aureus (MRSA), genome evolution of MRSA into VRSA, including strains engineered within the same patient under anti-staphylococcal therapy, may build up to future public health concern. To further complicate diagnosis, infection control and anti-microbial chemotherapy, non-sterile sites such as the nares and the skin could contain both S. aureus and coagulase-negative staphylococci (CoNS), either of which could harbour mecA the gene driving staphylococcal methicillin-resistance and required for MRSA-VRSA evolution. Results A new heptaplex PCR assay has been developed which simultaneously detects seven markers for: i) eubacteria (16S rRNA), ii) Staphylococcus genus (tuf), iii) Staphylococcus aureus (spa), iv) CoNS (cns), v) Panton-Valentine leukocidin (pvl), vi) methicillin resistance (mecA), and vii) vancomycin resistance (vanA). Following successful validation using 255 reference bacterial strains, applicability to analyse clinical samples was evaluated by direct amplification in spiked blood cultures (n = 89) which returned 100 % specificity, negative and positive predictive values. The new assay has LoD of 1.0x103 CFU/mL for the 16S rRNA marker and 1.0x104 CFU/mL for six other markers and completes cycling in less than one hour. Conclusion The speed, sensitivity (100 %), NPV (100 %) and PPV (100 %) suggest the new heptaplex PCR assay could be easily integrated into a routine diagnostic microbiology workflow. Detection of the cns marker allows for unique identification of CoNS in mono-microbial and in poly-microbial samples containing mixtures of CoNS and S. aureus without recourse to the conventional elimination approach which is ambiguous. In addition to the SA-CoNS differential diagnostic essence of the new assay, inclusion of vanA primers will allow microbiology laboratories to stay ahead of the emerging MRSA-VRSA evolution. To the best of our knowledge, the new heptaplex PCR assay is the most multiplexed among similar PCR-based assays for simultaneous detection of STAAR

Novel deletion alleles carrying CYP21A1P/A2 chimeric genes in Brazilian patients with 21-hydroxylase deficiency

<p>Abstract</p> <p>Background</p> <p>Congenital adrenal hyperplasia due to 21-hydroxylase deficiency is caused by deletions, large gene conversions or mutations in <it>CYP21A2 </it>gene. The human gene is located at 6p21.3 within a <it>locus </it>containing the genes for putative serine/threonine Kinase <it>RP</it>, complement <it>C4</it>, steroid 21-hydroxylase <it>CYP21 </it>tenascin <it>TNX</it>, normally, in a duplicated cluster known as RCCX module. The <it>CYP21 </it>extra copy is a pseudogene (<it>CYP21A1P</it>). In Brazil, 30-kb deletion forming monomodular alleles that carry chimeric <it>CYP21A1P/A2 </it>genes corresponds to ~9% of disease-causing alleles. Such alleles are considered to result from unequal crossovers within the bimodular <it>C4/CYP21 locus</it>. Depending on the localization of recombination breakpoint, different alleles can be generated conferring the locus high degree of allelic variability. The purpose of the study was to investigate the variability of deleted alleles in patients with 21-hydroxylase deficiency.</p> <p>Methods</p> <p>We used different techniques to investigate the variability of 30-kb deletion alleles in patients with 21-hydroxylase deficiency. Alleles were first selected after Southern blotting. The composition of <it>CYP21A1P/A2 </it>chimeric genes was investigated by ASO-PCR and MLPA analyses followed by sequencing to refine the location of recombination breakpoints. Twenty patients carrying at least one allele with <it>C4/CYP21 </it>30-kb deletion were included in the study.</p> <p>Results</p> <p>An allele carrying a <it>CYP21A1P/A2 </it>chimeric gene was found unusually associated to a <it>C4B/C4A </it><it>Taq </it>I 6.4-kb fragment, generally associated to <it>C4B </it>and <it>CYP21A1P </it>deletions. A novel haplotype bearing both p.P34L and p.H62L, novel and rare mutations, respectively, was identified in exon 1, however p.P30L, the most frequent pseudogene-derived mutation in this exon, was absent. Four unrelated patients showed this haplotype. Absence of p.P34L in <it>CYP21A1P </it>of normal controls indicated that it is not derived from pseudogene. In addition, the combination of different approaches revealed nine haplotypes for deleted 21-hydroxylase deficiency alleles.</p> <p>Conclusions</p> <p>This study demonstrated high allelic variability for 30-kb deletion in patients with 21-hydroxylase deficiency indicating that a founder effect might be improbable for most monomodular alleles carrying <it>CYP21A1P/A2 </it>chimeric genes in Brazil.</p

Adjusted Wallace Coefficient as a Measure of Congruence between Typing Methods ▿

We propose a new coefficient, the adjusted Wallace coefficient (AW), and corresponding confidence intervals (CI) as quantitative measures of congruence between typing methods. The performance of the derived CI was evaluated using simulated data. Published microbial typing data were used to demonstrate the advantages of AW over the Wallace coefficient