Clinical and mutational spectrum of Colombian patients with Pelizaeus Merzbacher Disease / Heterogeneidad clínica y mutacional en pacientes colombianos con Pelizaeus Merzbacher

Abstract Case Presentation: Pelizaeus Merzbacher Disease (PMD) is an X-linked developmental defect of myelination that causes childhood chronic spastic encephalopathy. Its genetic etiology can be either a duplication (or other gene dosage alterations) or a punctual mutation at the PLP1 locus. Clinically, it presents with developmental delay, nystagmus and, spasticity, supported by neuroimaging in which the defect of myelination is evident. We present a series of seven Colombian patients diagnosed with this leucodystrophy, describing their genotypic and phenotypic characteristics and heterogeneity. Clinical Findings: All patients included were male, 6 months to 16 years of age. Mean age at onset of symptoms was 8 months. Mean age at diagnosis was 5 years 5 months, being classic PMD most frequently diagnosed, as compared to the connatal phenotype. All cases had a primary diagnosis of developmental delay on 100%, and in 28.7% of cases, early onset nystagmus was described. 85.7% of patients had spasticity, 71.4% cerebellar signs, 57.0% hypotonia, and 28.5% had an abnormal movement disorder. Only three patients were able to achieve gait, though altered. In the two patients who had a diagnosis of connatal PMD maturational ages in danger zones according to the WHO Abbreviated Scale of Psychosocial Development were documented. All cases had abnormalities in neuroimages. Molecular Analysis and Results: Molecular studies were used in the majority of the cases to confirm the diagnosis (85.7 %). For two cases molecular confirmation was not considered necessary given their affected male brothers had already been tested. PLP1 gene dosage alterations (duplications) were found in 28.5 % of the patients (two siblings), whereas three different single nucleotide variants were detected. Clinical Relevance: According to these findings, as authors we propose the diagnostic algorithm in Colombian population to begin on a high clinical suspicion, followed by paraclinical extension, moving on to the molecular confirmation by using approaches to simultaneously sequence the PLP1 gene in order to detect point mutations and in/dels and performing a copy number variation analysis for the detection of gene dosage alterations.

Resumen Descripción del caso: La enfermedad de Pelizaeus Merzbacher es una leucodistrofia ligada al X que causa encefalopatía espástica crónica en la infancia. Su etiología es genética, por duplicaciones u otros trastornos de la dosis génica o mutaciones puntuales del gen PLP1, lo que condiciona la formación anormal de las vainas de mielina principalmente en el sistema nervioso central. Clínicamente se caracteriza por un cuadro de retardo del neurodesarrollo, nistagmus y espasticidad, con neuroimágenes que evidencian la dismielinización. Presentamos una serie de siete casos colombianos con esta leucodistrofica en la que describimos fenotípica y genotípicamente la heterogeneidad de esta enfermedad en nuestra población. Hallazgos clínicos: Todos los pacientes analizados fueron de sexo masculino, con edad promedio de inicio de síntomas hacia los ocho meses de vida. La edad media al diagnóstico fue de 5 años 5 meses, siendo más frecuente el diagnóstico de PMD clásica que el tipo connatal. Se describe retardo del desarrollo motor en el 100% de los casos, acompañado de nistagmus en el 28.7%. 85.7% de los casos tenía algún grado de espasticidad, 71.4 % signos cerebelosos, 57.0% hipotonía, y hasta en 28.5% se evidenciaron movimientos anormales. Solo tres pacientes lograron marcha, aunque patológica. En los dos pacientes con la forma connatal se documentó una edad maduracional motora en el rango de Alerta, de acuerdo a la escala abreviada del desarrollo de la OMS. En todos los casos se detectó algún tipo de anormalidad en el estudio imagenológico cerebral. Estudios Moleculares y Resultados: El diagnóstico molecular se empleó en la mayoría de los casos (85.7%), encontrando alteraciones en la dosis génica en el 28.5% y tres diferentes mutaciones puntuales. Relevancia clínica: Dados los hallazgos en los resultados del estudio molecular, sugerimos que en el abordaje diagnóstico confirmatorio para la población colombiana se debería contemplar en un mismo tiempo tanto la secuenciación como el estudio de variantes del número de copias del gen afectado, contrario a lo sugerido en literatura mundial en la que se inicia con estudio para duplicación / deleción.

Higher prevalence and gene amplification of HPV16 in oropharynx as compared to oral cavity

ABSTRACT Objective The objective of this study was to clarify differences regarding HPV16 infection and gene amplification between the oral cavity and oropharynx in healthy individuals. Material and Methods The subjects were 94 healthy asymptomatic individuals (41 males, 53 females; mean age 58.6 years, range 16-97 years) who visited the Department of Oral and Maxillofacial Reconstructive Surgery of the Hiroshima University Hospital from 2014 to 2015. Oral epithelial cells were collected from oral rinse and pharynx gargle samples and placed in saline. The human endogenous retrovirus gene ERV3-1 was used as a reference to estimate the number of human cells in each sample. DNA samples were extracted from approximately 10,000 human cells and tested for HPV16 DNA by PCR using a type-specific primer. Similarly, we analyzed the HPV16 viral copy number in HPV16-positive cases using real-time PCR to examine genomic amplification. Results The percentage of HPV16-positive cases was higher in the gargle (28.7%) as compared to the rinse (16.0%) samples. In the oral rinse samples, males (26.8%) showed a significantly higher rate of HPV16 than females (7.5%) (P=0.021). Importantly, in older subjects (aged 60-89 years), gargle samples showed a significantly higher rate of HPV16 (33.3%) than oral rinse samples (13.7%) (P=0.034). The average number of viral copies was approximately 8 times higher in the gargle than in the oral rinse samples (0.16±0.27 vs. 1.35±1.26 copy numbers per cell), a significant difference (P<0.001). Conclusion Our findings suggest that the oropharynx is more susceptible to HPV16 infection as compared to the oral cavity, while HPV16 gene amplification is also more commonly found in the oropharynx.

Investigação citogenômica tecidual post-mortem em portadores de malformações congênitas / Post-mortem tissue cytogenomics investigation in patients with congenital malformations

Introdução: As malformações congênitas (MCs) são a segunda causa de mortes fetais e infantis no Brasil e, em grande parte dos casos, a sua etiologia não é bem definida. Devido às consequências clínicas das MCs, alguns pacientes falecem sem tempo hábil para uma investigação etiológica acurada. Dessa forma, a maioria dos casos permanece sem uma confirmação molecular das suspeitas clínicas, dificultando o aconselhamento genético para as famílias. Objetivos: O presente trabalho utilizou técnicas citogenômicas para caracterizar molecularmente a presença de anormalidades no DNA, desde aneuploidias até a variação do número de cópias gênicas (CNVs) em diferentes tecidos de pacientes falecidos portadores de MC encaminhados ao Serviço de Verificação de Óbitos para avaliação anatomopatológica. Casuística e Métodos: Foram avaliadas amostras de 30 pacientes portadores de MC submetidos à necropsia. O DNA foi extraido de diferentes tecidos (cérebro, coração, fígado, pele e diafragma) previamente conservados em RNA later, formol ou emblocados em parafina. Foram utilizadas as técnicas de Multiplex Ligation-dependent Probe Amplification (MLPA) com os kits P095, P064 e P070 (MRC-Holland®), Marcadores Microssatélites (MMS) com o kit MiniFiler (Life Technologies®), a Fluorescence in Situ Hybridization (FISH), a técnica de array (Infinium® CytoSNP-850K BeadChip - Illumina) e o Sequenciamento Bidirecional por Sanger. A interpretação dos resultados foi realizada utilizando os softwares GeneMarker, Coffalyser, BlueFuse Multi, Sequencher e com os bancos de dados Database of Genomic Variants (DGV - http://projects.tcag.ca/variation/), Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources (DECIPHER - http://decipher.sanger.ac.uk/), UCSC Genome Bioinformatics (http://genome.ucsc.edu) e Mutation Taster. Resultados: Dos 30 pacientes avaliados, 13 apresentaram alterações patogênicas. Entre eles, oito apresentaram aneuploidias envolvendo...

Introduction: Congenital malformations (CMs) are the second leading cause of fetal and infant deaths in Brazil and in most cases the etiology is not well defined. Also, the patients remain without a conclusive diagnostic making difficult the genetic counseling. Objectives: This study applied cytogenomics techniques in order to characterize the presence of DNA abnormalities, as well as, aneuploidies and genomic copy number variations (CNVs) in different tissues from deceased patients with CM from "Serviço de Verificação de Óbitos". Patients and Methods: We evaluated samples from 30 patients undergoing necropsy. The DNA was extracted from different tissues (brain, heart, liver, skin and diaphragm) stored in RNA later, formaldehyde and embedded in paraffin. We performed Multiplex Ligation-dependent Probe Amplification (MLPA) with P095 kits, P064 and P070 (MRC-Holland®), microsatellite markers (MMS) with MiniFiler kit (Life Technologies), Fluorescence In Situ Hybridization (FISH), array technique (Infinium® CytoSNP-850K BeadChip - Illumina) and bidirectional sequencing by Sanger. The results was analyzed using different softwares: GeneMarker, Coffalyser, BlueFuse Multi Sequencher and databases Database of Genomic Variants (DGV - http://projects.tcag.ca/variation/) Database of Chromosomal Imbalance and Phenotype in Humans Using Ensembl Resources (Decipher - http://decipher.sanger.ac.uk/), UCSC Genome Bioinformatics (http://genome.ucsc.edu) and Mutation Taster. Results: The results showed 13 patients with pathogenic CNVs, and among them, eight presented aneuploidies involving chromosomes 13, 18, 21, X and Y. Two of them presented intra-tissue mosaicism. Also four patients showed several different microdeletions or microduplications: duplication of TYMS gene (18p11.32); deletion of CHL1 gene (3p26.3); deletion of HIC1 gene (17p13.3); deletion of TOM1L2 gene (17p11.2 ). One patient showed a pathogenic missense mutation of g.8535C>G (c.746C > G) in exon 7 from FGFR3...

eNOS se correlaciona com a biogênese mitocondrial em corações com cardiopatia congênita e cianose / eNOS correlates with mitochondrial biogenesis in hearts of congenital heart disease with cyanosis

FUNDAMENTO: O programa de biogênese mitocondrial no coração parece apresentar remodelação adaptativa após estresse biomecânico e oxidativo. Os mecanismos adaptativos que protegem o metabolismo do miocárdio durante a hipóxia são coordenados, em parte, pelo óxido nítrico (NO). OBJETIVO: Observar a biogênese mitocondrial e expressão do óxido nítrico sintase (NOS) em corações de cardiopatia congênita com cianose; discutir a resposta mitocondrial à hipóxia crônica do miocárdio. MÉTODOS: Foram investigados 20 pacientes com defeitos cardíacos cianóticos (n = 10) ou acianóticos (n = 10). Foram estudadas amostras do miocárdio na via de saída ventricular direita, tomadas durante a operação. A análise morfométrica de mitocôndrias foi realizada por microscopia eletrônica de transmissão. A relação mtDNA/nDNA foi determinada com PCR em tempo real. Os níveis de transcrição da subunidade I da citocromo c oxidase (COXI), coativador-1α do receptor γ ativado por proliferador de peroxissoma (PGC-1α), o fator respiratório nuclear 1 (NRF1), e fator de transcrição mitocondrial A (Tfam) foram detectados por reação em cadeia da polimerase via transcriptase reversa (RT-PCR) ativado por fluorescência em tempo real. Os níveis proteicos de COXI e nNOS, iNOS e eNOS foram medidos por técnica de Western Blot. RESULTADOS: A densidade volumétrica mitocondrial (Vv) e a densidade numérica (Nv) foram significativamente elevadas em pacientes com cianose, em comparação com a cardiopatia congênita acianótica. MtDNA elevada e suprarregulação dos níveis de COXI, PGC-1 α, NRF1 e Tfam mRNA foram observadas em pacientes cianóticos. Os níveis de proteína de COXI e eNOS foram significativamente maiores no miocárdio de pacientes cianóticos que nos de acianóticos. Os níveis de transcrição do PGC-1α se correlacionam com os níveis de eNOS. CONCLUSÃO: A biogênese mitocondrial é ativada no miocárdio da via de saída ventricular na cardiopatia congênita com cianose, que ...

BACKGROUND: Mitochondrial biogenesis program in heart appears to exhibit adaptive remodeling following biomechanical and oxidative stress. The adaptive mechanisms that protect myocardium metabolism during hypoxia are coordinated in part by nitric oxide (NO). OBJECTIVE: To observe mitochondrial biogenesis and nitric oxide synthase (NOS) expression in hearts of congenital heart disease with cyanosis, discuss mitochondrial response to chronic hypoxia in myocardium. METHODS: 20 patients with cyanotic (n=10) or acyanotic cardiac defects (n=10) were investigated. Samples from the right ventricular outflow tract myocardium taken during operation were studied. Morphometric analysis of mitochondria was performed with transmission electron microscope. Relative mtDNA/nDNA ratio was determined with real-time PCR. Cytochrome c oxidase subunit I (COXI), peroxisome-proliferator-activated receptor γ coactivator-1α (PGC-1α), nuclear respiratory factor 1 (NRF1), and mitochondrial transcription factor A (Tfam) transcript levels were detected by real-time fluorescent RT-PCR. COXI and nNOS, iNOS and eNOS protein levels were measured with western blot. RESULTS: Mitochondrial volume density (Vv) and numerical density (Nv) were significantly elevated in patients with cyanotic compared to acyanotic congenital heart disease. Elevated mtDNA and up-regulated COXI, PGC-1α, NRF1 and Tfam mRNA levels were observed in cyanotic patients. Protein levels of COXI and eNOS were significantly higher in the myocardium of cyanotic than of acyanotic patients. PGC-1α transcript levels correlated with the levels of eNOS. Conclusion: Mitochondrial biogenesis is activated in right ventricular outflow tract myocardium in congenital heart disease with cyanosis, which could be the adaptive response to chronic hypoxia and possibly involves eNOS up-regulation. (Arq Bras Cardiol. 2012; [online].ahead print, PP.0-0).