Espectro fenotípico de Síndrome de CHARGE neonatal / Phenotypic spectrum of neonatal CHARGE syndrome

INTRODUCCIÓN: El Síndrome de CHARGE (SCH), es un síndrome genético de amplia variabilidad fenotípica, de he rencia autosómica dominante, causado por variantes patogénicas en el gen CHD7. OBJETIVO: Descri bir el amplio espectro fenotípico de un SCH neonatal, heterocigoto para el gen CDH7 y la utilidad de la secuenciación en la confirmación diagnóstica, considerando los diagnósticos diferenciales. CASO CLÍNICO: recién nacida prematura de 34 semanas, con antecedentes prenatales de polihidroamnios severo, translucencia nucal aumentada y foco hiperecogénico cardiaco, con estudio de TORCH antenatal, que descartó infección congénita. Al nacer se pesquisó parálisis facial periférica, atresia de coanas, dismorfias múltiples, cardiopatía congénita y coloboma retinocoroideo bilateral. Las neuroimágenes mostraron hipoplasia de cóclea y de canales semicirculares bilaterales e hipoplasia pontocerebelosa. Los potenciales evocados auditivos mostraron hipoacusia sensorioneural profunda derecha y anacusia izquierda. Evolucionó con hipocalcemia y alteraciones en la inmunidad, confirmándose un hipoparatiroidismo e hipoplasia de timo. El cariograma fue normal y la amplificación de sondas dependiente de ligandos múltiples (MLPA) excluyó microdeleción 22q11.2. La sospecha clínica de SCH se confirmó con la detección de una variante patogénica en el gen CHD7. CONCLUSIONES: La su perposición de características clínicas del SCH con otros síndromes genéticos requiere confirmación genética molecular considerando diferencias en evolución, terapias y riesgos de recurrencia.

INTRODUCTION: CHARGE syndrome is a genetic disorder of wide phenotypic variability, of autosomal dominant in heritance, caused by pathogenic variants in the CHD7 gene. OBJECTIVE: To describe the broad pheno typic spectrum of neonatal CHARGE syndrome, heterozygous for the CHD7 gene, and the usefulness of genome sequencing in diagnostic confirmation, considering differential diagnoses. CLINICAL CASE: 34-week preterm newborn, with severe prenatal history of polyhydramnios, increased nuchal trans- lucency, and hyperechogenic cardiac focus, with a TORCH study that ruled out congenital infection. Peripheral facial paralysis, choanal atresia, multiple dysmorphisms, congenital heart disease, and bilateral retinochoroidal coloboma were observed at birth. The neuroimaging study showed hypo plasia of the cochlea and bilateral semicircular canals, and pontocerebellar hypoplasia. The auditory evoked potentials showed deep right-sided sensorineural hearing loss and left anacusis. The patient developed hypocalcemia and immunological alterations, confirming hypoparathyroidism and thy mus hypoplasia. The karyogram was normal and 22q11.2 microdeletion was excluded through mul tiplex ligation-dependent probe amplification (MPLA). A pathogenic variant in the CHD7 gene was detected that confirmed the clinical suspicion of CHARGE syndrome. CONCLUSIONS: The overlap of clinical characteristics of CHARGE syndrome requires molecular genetic confirmation, considering differences in evolution, therapies, and recurrence risks with other genetic syndromes.

Decreased expression of chromodomain helicase DNA-binding protein 9 is a novel independent prognostic biomarker for colorectal cancer

Previous studies suggested that chromodomain helicase DNA-binding proteins (CHDs), including CHD 1-8, were associated with several human diseases and cancers including lymphoma, liver cancer, colorectal cancer, stomach cancer, etc. To date, little research on CHD 9 in human cancers has been reported. In this study, we assessed the prognostic value of CHD 9 in patients with colorectal cancer (CRC). We screened for CHD 9 expression using immunohistochemical analysis in 87 surgical CRC specimens and found that the expression was upregulated in 81.5% of the cases, while 7.4% were decreased; in the remaining 11.1% of the cases, levels were not altered. Kaplan-Meier analysis showed that patients with high CHD 9 expression had better prognosis than those with low CHD 9 expression (54.5 vs 32.1%, P=0.034). Subsequently, Cox multi-factor survival regression analysis revealed that expression of CHD 9 protein was an independent predictor for CRC, with a hazard ratio of 0.503 (P=0.028). In addition, we found that CHD 9 expression was positively correlated with MSH2 (rs=0.232, P=0.036). We speculated that CHD9 might be a putative tumor suppressor gene, and could inhibit the development of CRC by participating in DNA repair processes. Our findings suggest that CHD 9 could be a novel prognostic biomarker and a therapeutic target for CRC. Further studies are needed to detect the effect of CHD 9 on cellular function and the expression of mismatch repair genes.

Caracterização termodinâmica e funcional da proteína não estrutural 3 (NS3) do vírus da dengue do tipo 2

O propósito deste trabalho foi a clonagem, expressão e purificação da proteína NS3 íntegra do DENV-2, seus domínios e complexos isoladamente, com objetivo de avaliar i) a estabilidade da NS3 íntegra e dos domínios protease e helicase; ii) o efeito do domínio protease sobre a atividade NTPásica; e iii) o papel do cofator NS2B na atividade e estrutura da NS3 protease. Utilizamos técnicas espectroscópicas para o monitoramento da fluorescência intrínseca do Trp e ensaios de atividade helicásica, ATPásica e proteásica. Nossos dados termodinâmicos demonstram a importância do domínio protease na estabilidade da NS3 íntegra, e que o interdomínio E169-K185 (que conecta os domínios protease e helicase) é importante na modulação da atividade proteolítica, interação da protease com o cofator NS2B, e estabilidade do domínio protease. Observamos que o cofator NS2B fornece maior estabilidade e cooperatividade ao domínio protease em pH básico, além de promover modificações conformacionais que reduzem a exposição de resíduos hidrofóbicos que parecem prejudicar na interação de resíduos dibásicos com a tríade catalítica. Ao avaliar se a baixa atividade ATPásica da NS3 íntegra em comparação com a NS3 helicase estaria associada com a oclusão do motivo Walker A pelo domínio protease, demonstramos que a NS3 helicase apresenta maior atividade ATPase, em pH 6,0, devido à exposição de resíduos hidrofóbicos que favorecem a ligação do ATP. Contudo, em pH fisiológico, a interação do ATP com o motivo Walker A foi maior na NS3 íntegra, demonstrando que a localização do domínio protease sobre o motivo de formação do complexo ATP-Mg2+ não interfere na interação. E finalmente, analisando a fenda de interação do ATP na NS3 íntegra dos 4 sorotipos do DENV, identificamos oito resíduos altamente conservados (R64, E66, G80, K201, R202, R209, K213 e R236) circundando esta região de ligação, que podem ser importantes no desenvolvimento de inibidores específicos desta enzima.

The aim of this work was the cloning, expression, and purification of the full-length NS3 protein of the DENV-2 as well as the its domains and protein complexes, aiming to study i) the thermodynamic stability of the full-length NS3, and of its protease and helicase domains; ii) the effect of the protease domain on ATPase activity; and iii) the effect of the NS2B cofactor on the structure and activity of the protease domain. We used fluorescence spectroscopy to monitor the Trp intrinsic fluorescence, and ATPase, helicase and protease activity assays. Our results thermodynamics showed the importance of the protease domain for the stabilization of the full-length NS3 protein of interdomain peptide (E169-K185) for activity, the interaction of the protease domain with cofactor NS2B, and the stability of the protease domain. We observed that the cofactor NS2B promoted not only a higher stability and cooperativity to the protease domain at basic pH, but also conformational changes that decreased the exposure of hydrophobic residues, disfavoring the interaction of dibasic residues with the catalytic triad. When we evaluated whether the decrease ATPase activity of the full-length NS3, in comparison with NS3 helicase, would be associated or not with the occlusion of the Walker A motif by protease domain, our results showed that the NS3 helicase increased ATPase activity at pH 6.0 because of the hydrophobic residue exposure favoring the ATP binding. On the other hand, the interaction of the ATP with Walker A motif was higher in the full-length NS3 than in the NS3helicase, suggesting that the localization of the protease domain on the helicase domain does not interfere with the interaction of the latter with the ATP-Mg+2. Finally, by analyzing the ATP binding site cleft in the full-length NS3 protein from the four DENV serotypes (DENV-1 to 4), we identified eight highly conserved residues (R64, E66, G80, K201, R202, R209, K213 and R236) surrounding this cleft that can be important in the development of specific inhibitors of that enzyme.

El nucléolo como un regulador del envejecimiento celular / The nucleolus as a regulator of cellular senescence

El nucléolo, considerado únicamente como el sitio de síntesis de los ribosomas, actualmente representa una estructura nuclear dinámica que participa en la regulación de importantes procesos celulares. Numerosas evidencias han demostrado que el envejecimiento celular es una de las diversas funciones que son controladas por el nucléolo. Las mutaciones en las proteínas de localización nucleolar promueven el envejecimiento prematuro en levaduras y humanos. La carencia de represión en la transcripción de genes que codifican para el ARNr que se encuentran dañados, y las mutaciones en las helicasas del ADN encargadas de minimizar la formación de círculos extra-cromosómicos del ADN que codifica para el ARNr, provocan modificaciones en la estructura del nucléolo e inducen envejecimiento prematuro en levaduras. De igual manera, en los humanos la carencia de las helicasas del ADN localizadas en el nucléolo y que participan en el mantenimiento de la integridad genómica, favorecen el desarrollo de aquellas enfermedades asociadas con el envejecimiento acelerado. Además, la presencia de algunos componentes de la telomerasa en el nucléolo, indica que parte de la biosíntesis de esta enzima se realiza en esta estructura nuclear, sugiriendo una conexión entre el nucléolo y la síntesis de los telómeros en la regulación del envejecimiento celular. Por otra parte, el nucléolo secuestra proteínas para regular su actividad biológica durante el inicio o término de la vida replicativa celular.

The nucleolus has been considered originally only as the site for the ribosome synthesis, but now it is well known that it represents a dynamic nuclear structure involved in important cellular processes. Several evidences have demonstrated that the nucleolus regulates the cellular senescence. Specific mutations on the DNAs codifying for nucleolar proteins induced premature senescence from yeast to human. The failure to repress the genes transcription codifying for damaged rRNA, and the mutations in DNA helicases, which minimizes the formation of DNA extra-chromosomal circles codifying for rRNA, modify the nucleolar structure and induce premature senescence in yeast. Similarly, in humans, the reduction of these DNA helicases levels, which are localized in the nucleoli and participate in maintenance of genomic integrity, helps to the development of those diseases associated with premature senescence. Furthermore, the presence in the nucleolus of some telomerase components, indicates that part of the biosynthesis of this enzyme occurred in this nuclear structure; suggesting a communication between the nucleolus and the synthesis of the telomeres in the regulation of cell senescence. On the other hand, the nucleolus sequesters proteins to regulate its own biological activity, from the start to the end of cellular replication. In addition this nuclear structure is involved in the biosynthesis of most cellular ribonucleoprotein particles, as well as in cell cycle regulation, making it central to gene expression. In conclusion, the nucleolus became a multifunctional subnuclear structure involved from cell proliferation to cell senescence.

Frequency of Werner helicase 1367 polymorphism and age-related morbidity in an elderly Brazilian population

Werner syndrome (WS) is a premature aging disease caused by a mutation in the WRN gene. The gene was identified in 1996 and its product acts as a DNA helicase and exonuclease. Some specific WRN polymorphic variants were associated with increased risk for cardiovascular diseases. The identification of genetic polymorphisms as risk factors for complex diseases affecting older people can improve their prevention, diagnosis and prognosis. We investigated WRN codon 1367 polymorphism in 383 residents in a district of the city of São Paulo, who were enrolled in an Elderly Brazilian Longitudinal Study. Their mean age was 79.70 ± 5.32 years, ranging from 67 to 97. This population was composed of 262 females (68.4 percent) and 121 males (31.6 percent) of European (89.2 percent), Japanese (3.3 percent), Middle Eastern (1.81 percent), and mixed and/or other origins (5.7 percent). There are no studies concerning this polymorphism in Brazilian population. These subjects were evaluated clinically every two years. The major health problems and morbidities affecting this cohort were cardiovascular diseases (21.7 percent), hypertension (83.7 percent), diabetes (63.3 percent), obesity (41.23 percent), dementia (8.0 percent), depression (20.0 percent), and neoplasia (10.8 percent). Their prevalence is similar to some urban elderly Brazilian samples. DNA was isolated from blood cells, amplified by PCR and digested with PmaCI. Allele frequencies were 0.788 for the cysteine and 0.211 for the arginine. Genotype distributions were within that expected for the Hardy-Weinberg equilibrium. Female gender was associated with hypertension and obesity. Logistic regression analysis did not detect significant association between the polymorphism and morbidity. These findings confirm those from Europeans and differ from Japanese population.