Novel deletions and unusual genetic mechanisms underlying alpha-thalassemia

Hemoglobin (Hb) is a protein responsible for oxygen transportation from lungs to the entire body. It is composed by four globular subunits - the globins - each with a central core containing a heme molecule. Globins are encoded by the α- and β-globin gene clusters located at 16p13.3 and 11p15.5, respectively. The pattern of globin genes expression during development is precisely controlled by the interaction of cis-regulatory genomic regions (located in close proximity to and far from genes) with trans-activating/silencing factors within permissive chromatin domains. In fact, approximately 25-65 kb upstream of the α-globin genes there are four multispecies conserved sequences (MCS-R1 to R4) which are critical for the expression regulation of the downstream globin genes. The main objectives of this work were to characterize the molecular lesions underlying eight unusual cases of α-thalassemia or Hb H disease, and to understand their origin and functional consequences. Deletions were detected by Multiplex Ligation-dependent Probe Amplification (MLPA) using the SALSA MLPA P140B HBA kit (MCR-Holland). Additionally, specifically designed synthetic MLPA probes, as well as Gap-PCR and Sanger sequencing were performed for fine deletion breakpoint mapping. We have found seven different deletions (ranging from 3.3 to ≈323 kb), four of them not previously described. The four largest deletions removed all the α-globin genes, whereas the other three deletions removed one or more of the distal regulatory elements keeping the globin genes structurally intact. In one case, only the MCS-R2 (also known as HS-40) was removed and replaced by a 39 nt DNA fragment possibly resulting from a complex rearrangement that introduces new pieces of DNA (probably from Chrs. 3 and 7) bridging the two deletion breakpoints. In the remaining case, no deletion was found and the patient revealed to be a very unusual case of acquired alpha-thalassemia-myelodysplastic syndrome. It is important to detect individuals with this type of uncommon deletions as there is a 25% risk of having a child with Hb Bart’s hydrops fetalis or Hb H disease if their partner is a carrier of an α0-thal or α+-thal allele, respectively. Moreover, further investigation is currently being developed on one of these natural mutants which is bringing new insights into the long-range regulation mechanism of the globin gene expression and to the pathophysiology of the α-thalassemia.N/

Alpha-thalassemia due to novel deletions and complex rearrangements in the subtelomeric region of chromosome 16p

2º Dia do Jovem Investigador do Instituto Nacional de Saúde Doutor Ricardo Jorge, INSA, 8 maio 2017Introduction: Inherited deletions removing the α-globin genes and/or their upstream regulatory elements (MCSs) give rise to alpha-thalassemia, one of the most common genetic recessive disorders worldwide. The pathology is characterized by microcytic hypochromic anemia due to reduction of the α-globin chain synthesis, which are essential for hemoglobin tetramerization. Material and Methods: In order to clarify the suggestive α-thalassemia phenotype in eleven patients, we performed Multiplex Ligation-dependent Probe Amplification with commercial and synthetic engineered probes, gap-PCR, and Sanger sequencing to search for deletions in the subtelomeric region of chromosome 16p. Results: We have identified five distinct large deletions, two of them novel, and one indel. The deletions range from approximately 3.3 to 323 kb, and i) remove the whole α-globin cluster; or ii) remove exclusively the upstream regulatory elements leaving the α-globin genes structurally intact. The indel consists in the loss of MCS-R2 (HS-40), which is the most important distal regulatory element for the α-globin gene expression, and the insertion of 39 bp, seemingly resulting from a complex rearrangement involving two DNA segments (probably from chromosome 3q) bridging the deletion breakpoints with a CC-bp orphan sequence in between. Finally, in one patient no α-globin deletion or point mutation were found. This patient revealed to be a very unusual case of acquired alpha-thalassemia associated with a myelodysplastic syndrome. Conclusions: Our study widens the spectrum of molecular lesions by which α-thalassemia may occur and emphasizes the importance of diagnosing large α-zero-deletions to provide patients with appropriate genetic counseling.info:eu-repo/semantics/publishedVersio

ADESÃO DOS IDOSOS COM DOENÇAS CRÔNICAS AO TRATAMENTO MEDICAMENTOSO / ADHERENCE OF THE ELDERLY WITH CHRONIC DISEASES TO MEDICAL TREATMENT

Introdução: As pessoas idosas podem ser acometidas por doenças e agravos crônicos não transmissíveis que requerem acompanhamento constante, pois, em razão da sua natureza, não têm cura e, frequentemente, estão associadas à comorbidades. Objetivo: Verificar a adesão medicamentosa dos idosos com condições crônicas. Métodos: Estudo descritivo com abordagem quantitativa realizado em um Centro de Saúde do município de São Luís (MA). A amostra foi de 308 idosos no período de dezembro de 2013 a junho de 2014. O questionário compreendia as condições crônicas apresentadas, as medicações utilizadas e os parâmetros psicométricos para a Medida de Adesão aos Tratamentos - MAT, analisado no programa software EPIINFO® versão 7.1.2. Resultados: Verificou-se que 30% dos idosos apresentaram hipertensão arterial sistêmica e 18% apresentaram diabetes mellitus e hipertensão arterial sistêmica, simultaneamente. O nível de adesão medicamentosa dos idosos com condições crônicas foi satisfatório, com94%. Conclusão: Concluiu-se que a adesão dos idosos ao tratamento medicamentoso contribui para a prevenção de complicações decorrentes das doenças crônicas, maior expectativa de vida e aumento da qualidade de vida.Palavras-chaves: Adesão à Medicação. Idoso. Doença crônica. Enfermagem.AbstractIntroduction: The older people can be affected by diseases and non-transmission chronic diseases that require constant monitoring, because, in reason of their nature, have no cure. These chronic conditions tend to manifest significantly in the elderly, and often are associated to comorbidities. Objective: Verify the adherence of elderly with chronic diseases to medical treatment. Methods: It is descriptive quantitative study conducted in a Health Center in São Luís, Maranhão, Brazil. The sample consisted of 308 elderly respondents in the period December 2012 to June 2013. The questionnaire comprised the chronic conditions presented, medications used and the psychometric parameters to Measure Treatment Adherence - MTA, analyzed the program EPIINFO® software version 7.1.2. Results: The results show that the group studied showed mainly the following chronic diseases: systemic arterial hypertension in 30% and diabetes mellitus and systemic arterial hypertension accounts for 18%, simultaneously. The level of medical adherence of elderly with chronic diseases was satisfactory, with 94%. Conclusion: It was concluded that the adherence of the elderly to medical treatment contributes to the prevention of complications of chronic diseases, increased life expectancy and improved quality of life.Keywords: Medication adherence. Elderly. Chronic disease. Nursing

Evaluación del nuevo Contador Hematológico Unicel ® DXH 800 – Beckman Coulter en el estudio citológico de los liquidos biológicos

Poster apresentado no LVII Congreso Nacional de la Sociedad Española de Hematología y Hemoterapia (SEHH)/ XXXI Congreso Nacional de la Sociedad Española de Trombosis y Hemostasia (SETH), 22-24 Outubro 2015, Valencia

Rare genetic variants involved in multisystem inflammatory syndrome in children: a multicenter Brazilian cohort study

IntroductionDespite the existing data on the Multisystem Inflammatory Syndrome in Children (MIS-C), the factors that determine these patients evolution remain elusive. Answers may lie, at least in part, in genetics. It is currently under investigation that MIS-C patients may have an underlying innate error of immunity (IEI), whether of monogenic, digenic, or even oligogenic origin.MethodsTo further investigate this hypothesis, 30 patients with MIS-C were submitted to whole exome sequencing. ResultsAnalyses of genes associated with MIS-C, MIS-A, severe covid-19, and Kawasaki disease identified twenty-nine patients with rare potentially damaging variants (50 variants were identified in 38 different genes), including those previously described in IFNA21 and IFIH1 genes, new variants in genes previously described in MIS-C patients (KMT2D, CFB, and PRF1), and variants in genes newly associated to MIS-C such as APOL1, TNFRSF13B, and G6PD. In addition, gene ontology enrichment pointed to the involvement of thirteen major pathways, including complement system, hematopoiesis, immune system development, and type II interferon signaling, that were not yet reported in MIS-C.DiscussionThese data strongly indicate that different gene families may favor MIS- C development. Larger cohort studies with healthy controls and other omics approaches, such as proteomics and RNAseq, will be precious to better understanding the disease dynamics

Nurse and midwife interventions to protect, promote and support breastfeeding: an umbrella review protocol

Objective: To synthesize the nurse and midwife interventions on protecting, promoting, and supporting breastfeeding. Introduction: Considering the benefits of breast milk, the WHO declares breastfeeding as a fundamental practice for infant health. Therefore, it recommends exclusive breastfeeding for all children, up to six months of age, and on a complimentary basis, up to at least two years of age. Despite this, about two out of every three babies under six months worldwide are not exclusively breastfed, representing a considerable divergence from the established goals. Worldwide breastfeeding rates are concerning for nurses and midwives involved in breastfeeding care, which raises questions about the knowledge and ability to promote it effectively. So, it makes it relevant to understand the nursing and midwifery intervention in the breastfeeding process, which justifies this umbrella review. Inclusion criteria: Will be considered all systematic review studies that explore the nurse and midwife intervention in protecting, promoting, and supporting breastfeeding in hospital and/or community settings. Methods: This umbrella review will be carried out according to the proposed methodology of the Joanna Briggs Institute (JBI). Published literature in English, Portuguese, and Spanish, from 2018 to the present will be included. Databases aggregated by the EBSCOhost, SCOPUS and Web of Science engine will be searched, including relevant grey literature sources. Two independent reviewers will extract data using a tool developed specifically for this umbrella review objective. The results will be presented in infographic form, accompanied by a narrative explanation of their relationship with the review questions

Novel and rare large deletions in the globin gene clusters causing different types of thalassemia

The major component of the red blood cells is hemoglobin A which consists of 2α- and 2β-globin chains encoded by α- and β-globin genes located in two different gene clusters (16p13.3 and 11p.15.5, respectively). Molecular defects (usually point mutation or short deletion) that give rise to a quantitative reduction of the corresponding globin chain, result in a hereditary hypochromic and microcytic anemia called thalassemia. However, rarely, the molecular basis of the pathology could be a large deletion affecting several globin genes and/or their distal regulatory sequence. Four patients with hematological phenotypes suggestive of thalassemia, in whom no globinic molecular abnormalities had been found by standard diagnostic procedures, were screened for deletions in the telomeric region of chromosome 16 and 11, by Multiplex Ligation-dependent Probe Amplification (MLPA) assay. To further characterize the breakpoints of the deletions found, we employed synthetic MLPA probemixes designed in our laboratory, as well as PCR and DNA sequencing. We identified two cases of α-thalassemia caused by two distinct large deletions which remove all α-like structural genes and their distal regulatory sites: both are telomeric, one presents at least 271.14 kb of length and the other, at least, 231 kb. Concerning β-globin cluster screening, two deletions were found: one has at least 186 kb, encloses the entire cluster and its locus control region, and gives rise to a εγδβ0-thalassemia. The other presents at least 3 kb, has its 5’ breakpoint located within the second intron of the β-globin gene and its 3’ end within the L1 repetitive region of the cluster. Both α- and β-cluster larger deletions are novel and were named --CMB/αα and PORTUGUESE εγδβ0-Thal, respectively. The other two smaller deletions, given the uncertainty regarding their breakpoints, might be similar to others already published. In all patients, genotypes are well correlated with the different thalassemic phenotypes presented. MLPA proves to be a useful technique to identify known and unknown large deletions affecting globin gene clusters.Trabalho parcialmente financiado por "Programa de financiamente plurianual do CIGMH"

Análise retrospetiva do desempenho dos participantes no Programa de Morfologia do Sangue Periférico - PNAEQ 2012-2017

O exame morfológico do sangue periférico é essencial para o diagnóstico clínico de doenças hematológicas e doenças não hematológicas com expressão morfológica no sangue periférico. A avaliação do desempenho dos profissionais deve ser uma prática implementada pelos laboratórios. A participação em programas de avaliação externa da qualidade, em que são enviadas amostras de doentes selecionadas pelos membros do Grupo de Trabalho de Hematologia do Programa Nacional de Avaliação Externa da Qualidade (PNAEQ), permite a avaliação e formação tendo como objetivo a melhoria do desempenho dos profissionais. No programa de Morfologia de Sangue Periférico do PNAEQ são enviados três ensaios anuais com três amostras cada. O Grupo de Trabalho de Hematologia foi formalizado em 2013 e conta com a colaboração de peritos nacionais, dos laboratórios de Centros Hospitalares Lisboa Norte e Ocidental e do Instituto Português de Oncologia de Lisboa, que contribuem voluntariamente com amostras de doentes, assim como com comentários complementares formativos. O objetivo deste trabalho é avaliar o desempenho dos participantes de laboratórios hospitalares e de ambulatório, no período 2012-2017, quanto à identificação das alterações morfológicas e hipóteses de diagnóstico[1] nas amostras esfregaços de sangue periférico.N/

External Quality Assessment in peripheral blood morphology - PNAEQ experience

Este poster ganhou o prémio de melhor poster apresentado.Peripheral blood morphology is a very important tool for the clinical diagnosis in haematology disorders. The National External Quality Assessment Program (PNAEQ), aims to promote EQA programs, using Expert Working Groups as methodology (WG). The Haematology WG was formalized in 2013 and has the collaboration of national experts, namely from hospital and oncology entities, who voluntarily contribute with samples and case study selection, as well as in monitoring and evaluation of the results, aiming to a continuous improvement of participants' performance. The aim of this work was the evaluation of participating hospital and ambulatory laboratories performance, in the period between 2015 and 2017, regarding the identification of the morphological alterations and diagnostic hypothesis(1) for the peripheral blood smear sent.N/