Investigation of the impact of AXL, TLR3, and STAT2 in congenital Zika syndrome through genetic polymorphisms and protein-protein interaction network analyses.

INTRODUCTION: Zika virus (ZIKV) is a human teratogen that causes congenital Zika syndrome (CZS). AXL, TLR3, and STAT2 are proteins involved in the ZIKV's entry into cells (AXL) and host's immune response (TLR3 and STAT2). In this study, we evaluated the role of genetic polymorphisms in these three genes as risk factors to CZS, and highlighted which proteins that interact with them could be important for ZIKV infection and teratogenesis. MATERIALS AND METHODS: We evaluate eighty-eight children exposed to ZIKV during the pregnancy, 40 with CZS and 48 without congenital anomalies. The evaluated polymorphisms in AXL (rs1051008), TLR3 (rs3775291), and STAT2 (rs2066811) were genotyped using TaqMan® Genotyping Assays. A protein-protein interaction network was created in STRING database and analyzed in Cytoscape software. RESULTS: We did not find any statistical significant association among the polymorphisms and the occurrence of CZS. Through the analyses of the network composed by AXL, TLR3, STAT2 and their interactions targets, we found that EGFR and SRC could be important proteins for the ZIKV infection and its teratogenesis. CONCLUSION: In summary, our results demonstrated that the evaluated polymorphisms do not seem to represent risk factors for CZS; however, EGFR and SRC appear to be important proteins that should be investigated in future studies.

Contribution of miR-124 rs531564 polymorphism to the occurrence of congenital Zika syndrome.

Zika virus (ZIKV) cause Congenital Zika Syndrome (CZS) in individuals exposed during pregnancy. Studies have shown that ZIKV infection positively regulates the miR-124 expression in neural cells, which leads to a decrease of TFRC, a gene targeted of this miRNA. Both miR-124 and TFRC exhibit a pivotal role in nervous system development. Therefore, in this study we aimed to investigate whether genetic variants that affect the expression of these genes could act together with ZIKV to increase the risk of individuals developing CZS. TFRC rs406271 and MIR-124-1 rs531564 polymorphisms were genotyped, using TaqMan® Genotyping Assays, in a sample of children who were exposed to ZIKV during pregnancy, of whom 40 were born with CZS and 48 without congenital anomalies. We identified that individuals with CZS presented a higher frequency of CG genotype of rs531564 polymorphism in MIR-124-1 (p=0.048), which is associated with increased expression of miR-124. Since ZIKV also upregulates the expression of this miRNA, the presence of CG genotype in individuals exposed to the virus could lead to a scenario of overexpression of miR-124 in the brain. Since teratogenesis is a multifactorial event, this genetic finding could partly explain why such individuals are more susceptible to CZS, considering both the downregulation of important neurodevelopment genes, as well as deregulation of the neurogenesis process. Thus, we provide preliminary evidence about a possible genetic risk factor to CZS and highlight the importance of analyzing functional polymorphisms related to epigenetic modulators of neurodevelopment genes in the context of ZIKV teratogenesis.

Functional Polymorphisms in the p53 Pathway Genes on the Genetic Susceptibility to Zika Virus Teratogenesis.

Congenital Zika Syndrome (CZS) occurs in up to 42% of individuals exposed to ZIKV prenatally. Deregulation in gene expression and protein levels of components of the p53 signaling pathway, such as p53 and MDM2, due to ZIKV infection has been reported. Here, we evaluate functional polymorphisms in genes of the p53 signaling pathway as risk factors to CZS. Forty children born with CZS and forty-eight children exposed to ZIKV, but born without congenital anomalies were included in this study. Gestational and sociodemographic information as well as the genotypic and allelic frequencies of functional polymorphisms in TP53, MDM2, MIR605 and LIF genes were compared between the two groups. We found children with CZS exposed predominantly in the first trimester and controls in the third trimester (p<0.001). Moreover, children with CZS were predominantly from families with a lower socioeconomic level (p=0.008). We did not find a statistically significant association between the investigated polymorphisms and development of CZS; however, by comparing individuals with CZS and lissencephaly or without lissencephaly, we found a significative difference in the allelic frequencies of the TP53 rs1042522, which is associated with a more potent p53-induced apoptosis (p=0.007). Our findings suggest that the TP53 rs1042522 polymorphism should be better investigate as a genetic risk factor for the development of lissencephaly in children with CZS.

Association between Genetic Variants in NOS2 and TNF Genes with Congenital Zika Syndrome and Severe Microcephaly.

Zika virus (ZIKV) causes Congenital Zika Syndrome (CZS) in individuals exposed prenatally. Here, we investigated polymorphisms in VEGFA, PTGS2, NOS3, TNF, and NOS2 genes as risk factors to CZS. Forty children with CZS and forty-eight children who were in utero exposed to ZIKV infection, but born without congenital anomalies, were evaluated. Children with CZS were predominantly infected by ZIKV in the first trimester (p < 0.001) and had mothers with lower educational level (p < 0.001) and family income (p < 0.001). We found higher risk of CZS due the allele rs2297518[A] of NOS2 (OR = 2.28, CI 95% 1.17-4.50, p = 0.015). T allele and TT/CT genotypes of the TNF rs1799724 and haplotypes associated with higher expression of TNF were more prevalent in children with CZS and severe microcephaly (p = 0.029, p = 0.041 and p = 0.030, respectively). Our findings showed higher risk of CZS due ZIKV infection in the first trimester and suggested that polymorphisms in NOS2 and TNF genes affect the risk of CZS and severe microcephaly.

Alterações eritrocitárias em pacientes com Lupus Eritematoso Sistêmico / Erythrocytic changes in patients with Systemic Lupus Erythematosus

Objetivo: Determinar as alterações eritrocitárias em pacientes com Lupus Eritematoso Sistêmico (LES), atendidos no Hospital Geral Universitário (HGU) Cuiabá/MT. Métodos: Amostras de sangue de 40 pacientes, do sexo feminino foram coletadas e processadas (automação ABX Pentra 80 – citometria defluxo). As análises estatísticas descritivas foram feitas com auxílio do software estatístico IBM SPSS 2.0. Resultados: Dos 40 pacientes analisados, 42,5% apresentaram anemia. As médias dos valores de hematócrito e dosagem de hemoglobina nos pacientes com anemia foram respectivamente de 29,7% e 9,9 g/dL, ao passo que naqueles nos quais não se observou quadro anêmico a média de hematócrito foide 41,1% e a de dosagem de hemoglobina 13,7g/dL. A anemia normocítica normocrômica foi a mais encontrada (64,7%) sendo observados, nessa situação, índices hematimétricos (VCM, HCM, CHCM e RDW) dentro dos valores de normalidade. As anemias macrocíticas e microcíticas tiveram a mesma representação percentual (17,6%). Entre as anemias macrocíticas, alterações hematimétricas, tais como, RDW aumentado, policromasia e reticulocitose observadas em alguns pacientes sugerem a possibilidade de processos hemolíticos comumente observados nessa patologia. Conclusão: Evidencia-se a necessidade de avaliar cuidadosa e frequentemente os achados do hemograma, pois é uma ferramenta importante na avaliação do paciente e da eficácia do tratamento.

Objective: We assessed the prevalence of erythrocytic changes in patients with SLE, at Cuiabá University General Hospital (UNIC-HGU). Methods: blood samples from 40 female patients were collected and processed (ABX Pentra 80 automation – flow cytometry). Descriptive statistical analyses were performed using IBM SPSS 2.0 statistical software. Results: 42.5% of the 40 patients showed lowered concentrations of hemoglobin. The hematocrit and hemoglobin dosage averages of the anemic patients were respectively of 29.7% and 9.9 gdL, while those without anemia showed 41.1% of hematocrit average and hemoglobin average dosage of 13,7gdL. Normocytic and normochromic anemia was the most prevalent, founded in 64.7% of the anemic patients, with normal hematimetric indices. In cases of macrocytic anemia, hematimetric changes, such as increased RDW, policromasy and reticulocytosis observed in some patients suggest hemolytic processes, commonly observed in these patients. Conclusion: Anemia was found in 42% of the patients and careful observation of the erythrocytic changes is an important tool in the follow up of LES patients.

Ethical issues related to the access to orphan drugs in Brazil: the case of mucopolysaccharidosis type I.

BACKGROUND/AIMS: Mucopolysaccharidosis type I (MPS I) is a rare lysosomal storage disorder treated with bone marrow transplantation or enzyme replacement therapy with laronidase, a high-cost orphan drug. Laronidase was approved by the US Food and Drug Administration and the European Medicines Agency in 2003 and by the Brazilian National Health Surveillance Agency in 2005. Many Brazilian MPS I patients have been receiving laronidase despite the absence of a governmental policy regulating access to the drug. Epidemiological and treatment data concerning MPS I are scarce. This study aims to present a demographic profile of Brazilian patients with MPS I, describe the routes of access to laronidase in Brazil, and discuss associated ethical issues relating to public funding of orphan drugs. METHODS: In this cross-sectional observational study, data were collected nationwide between January and September 2008 from physicians, public institutions and non-governmental organisations involved with diagnosis and treatment of MPS I, using two data collection instruments specifically designed for this purpose. RESULTS: The minimum prevalence of MPS I in Brazil was estimated at 1/2,700,000. Most patients (69.8%) were younger than 15 years; 60 (88.2%) received laronidase. The most common route of access to the drug was through lawsuits (86.6%). CONCLUSIONS: In Brazil, MPS I is predominantly a paediatric illness. Even though the cost of laronidase treatment is not officially covered by the Brazilian government, most MPS I patients receive the drug, usually through litigation. This gives rise to major ethical conflicts concerning drug access in a low-resource context. The Brazilian health policy framework lacks evidence-based clinical protocols for the distribution of orphan drugs.

Molecular identification of chromosome Y sequences in Brazilian patients with Turner syndrome.

The investigation of Y-specific sequences in patients with Turner Syndrome (TS) with karyotype 45,X or mosaic, has a fundamental role in the clinical management of these patients. The relationship between the presence of Y chromosome fragments and a higher risk of gonadoblastoma in TS has already been established. The aim of the study was to investigate the presence of Y-chromosome fragments in a population of 42 female Brazilian patients with TS from Mato Grosso state. Cytogenetic analysis has shown the karyotypes 45,X in 27 of them (64.3%) and mosaic in 15 (35.7%). The presence of the Y-primers SRY, DYZ3, ZFY, DYZ1, DYS1 and PABY was investigated in all patients. These markers were amplified by polymerase chain reaction (PCR) technique, using DNA genomic from peripheral blood lymphocytes. None of these patients had shown any Y-chromosome fragments when they were analysed only by the classic cytogenetic technique. The PCR analysis with the Y-specific sequences ZFY and DYZ3 were identified in two different patients (4.8%), both with karyotype 45,X. It was concluded that PCR is efficient in the investigation of hidden Y-fragments in TS patients. Therefore, this method should be included in the routine assistance of these patients.

Deletion of 17p13 and LIS1 gene mutation in isolated lissencephaly sequence.

Classical lissencephaly is a neuroblast migration disorder that occurs either as isolated lissencephaly sequence or in association with malformation syndromes, such as the Miller-Dieker syndrome. In this work, alterations of the LIS1 gene in patients diagnosed as having isolated lissencephaly sequence were investigated. Ten patients were evaluated for the following aspects: classical cytogenetics by karyotyping using solid staining and G-banding; molecular cytogenetics using fluorescent in situ hybridization with a specific probe for the critical region of isolated lissencephaly sequence; and molecular analysis using deoxyribonucleic acid sequencing. Classical cytogenetic analysis indicated apparently normal karyotypes in all patients, but fluorescent in situ hybridization revealed a 17p13.3 microdeletion in one. In another patient, deoxyribonucleic acid sequencing disclosed a 1 base pair insertion in exon 4 within a sequence of eight consecutive adenine residues (162-163insA), a mutation that predicts a truncated protein. Two different polymorphisms were also detected: a T>C substitution in intron 6 (c.568 + 27bp T>C) and a C>T substitution in the nontranslated region of exon 11 (1250 C>T). These results indicate that cytogenetic analysis and molecular investigation of the LIS1 gene are not always sufficient to determine the disease etiology. These findings are consistent with previous studies and suggest the involvement of other genes in cortical malformation.

Evaluation of a protocol for postmortem examination of stillbirths and neonatal deaths with congenital anomalies.

A study was conducted on 75 perinatal deaths with congenital anomalies through clinical, radiographic, cytogenetic, and autopsy evaluation, and the diagnoses of 72 patients (96%) were determined. In 11 patients with chromosomal anomalies, the cytogenetic study was sufficient to determine the diagnosis and the reproductive risk. In these cases, the value of the autopsy results resided above all in the description of the clinical variability. Radiographic evaluation was the best method to establish a diagnosis of skeletal dysplasias (14.7%). Furthermore, the X-rays showed small skeletal defects which are difficult to see on dissection. The clinical genetic evaluation with a detailed description of the phenotype and anthropometric exam, performed by a clinical geneticist, and the autopsy with gross and microscopic evaluation, facilitated the diagnoses of 50 cases (66.7%). We concluded that, in perinatal death with congenital anomalies, the teamwork of clinical geneticists and fetal pathologists increases the probability of determining the etiological diagnosis. This is essential to define the parents' reproductive risk, thus contributing to primary prevention of congenital anomalies.

Ceratocone e retardo mental entre irmäos / Keratoconus and mental retardation among siblings

Apresentamos dois irmãos com a associação de retardo mental e ceratocone bilateral, filhos de pais consagüíneos, onde a avaliação genealógica sugere um padrão de herança autossômica recessiva. Uma nova síndrome monogênica pode ser sugerida. Um dos pacientes foi submetido a transplante de córnea penetrante e evoluiu com úlcera corneana infecciosa, com boa resposta ao tratamento clínico. Bons resultados podem ser obtidos se a indicação de transplante de córnea em retardados mentais for criteriosa.