Increased DNA Copy Number Variation Mosaicism in Elderly Human Brain.

Aging is a complex process strongly determined by genetics. Previous reports have shown that the genome of neuronal cells displays somatic genomic mosaicism including DNA copy number variations (CNVs). CNVs represent a significant source of genetic variation in the human genome and have been implicated in several disorders and complex traits, representing a potential mechanism that contributes to neuronal diversity and the etiology of several neurological diseases and provides new insights into the normal, complex functions of the brain. Nonetheless, the features of somatic CNV mosaicism in nondiseased elderly brains have not been investigated. In the present study, we demonstrate a highly significant increase in the number of CNVs in nondiseased elderly brains compared to the blood. In two neural tissues isolated from paired postmortem samples (same individuals), we found a significant increase in the frequency of deletions in both brain areas, namely, the frontal cortex and cerebellum. Also, deletions were found to be significantly larger when present only in the cerebellum. The sizes of the variants described here were in the 150-760 kb range, and importantly, nearly all of them were present in the Database of Genomic Variants (common variants). Nearly all evidence of genome structural variation in human brains comes from studies detecting changes in single cells which were interpreted as derived from independent, isolated mutational events. The observations based on array-CGH analysis indicate the existence of an extensive clonal mosaicism of CNVs within and between the human brains revealing a different type of variation that had not been previously characterized

Fetal sex determination in twin pregnancies using non-invasive prenatal testing

Non-invasive prenatal testing (NIPT) is accurate for fetal sex determination in singleton pregnancies, but its accuracy is not well established in twin pregnancies. Here, we present an accurate sex prediction model to discriminate fetal sex in both dichorionic diamniotic (DCDA) and monochorionic diamniotic/monochorionic monoamniotic (MCDA/MCMA) twin pregnancies. A retrospective analysis was performed using a total of 198 twin pregnancies with documented sex. The prediction was based on a multinomial logistic regression using the normalized frequency of X and Y chromosomes, and fetal fraction estimation. A second-step regression analysis was applied when one or both twins were predicted to be male. The model determines fetal sex with 100% sensitivity and specificity when both twins are female, and with 98% sensitivity and 95% specificity when a male is present. Since sex determination can be clinically important, implementing fetal sex determination in twins will improve overall twin pregnancies management

Genomic and epigenomic alterations in the anatomopathological and cognitive manifestations of Alzheimer\'s disease

A doença de Alzheimer (DA) é a causa mais comum de demência na população, sendo responsável por cerca de 50 a 60% dos casos. Embora o diagnóstico clínico da doença na maioria das vezes seja acurado, a confirmação da DA só é feita post mortem através principalmente da caracterização dos dois tipos principais de lesões neurais: depósitos extracelulares de placas de β amiloide e emaranhados de proteína tau hiperfosforilada. Até o momento, o envolvimento de apenas quatro genes foi confirmado na etiologia da DA, três deles (APP, PSEN1 e PSEN2) associados à forma familial de herança mendeliana, que corresponde a um tipo raro e grave. No entanto, apesar de inúmeros trabalhos de associação genômica, (Genome wide association studies- GWAS) sugerirem uma possível participação de vários outros genes na suscetibilidade à manifestação da forma multifatorial da DA, o gene APOE, ainda é o único consistente e reproduzivelmente associado à doença. As descobertas derivadas dos GWAS investigando o papel de SNPs coletivamente explicam somente uma pequena porcentagem da variação herdada que contribui para o risco de desenvolver a DA. Atualmente, há novas abordagens para investigar a base genética do restante da variabilidade fenotípica herdada e que pode influenciar a suscetibilidade ao desenvolvimento de doenças complexas. O papel da variação do número de cópias de segmentos de DNA (Copy Number Variation - CNV) na genética de doenças complexas foi demonstrado por diversos estudos nos últimos anos e evidencia que desequilíbrios genômicos também podem contribuir significantemente para a resistência ou susceptibilidade a várias patologias. Outro aspecto que vem assumindo crescente importância é a análise de modificações epigenéticas que podem constituir um mecanismo molecular básico e contribuir diretamente para a patogênese da DA. Logo, este trabalho teve como objetivo principal investigar dois aspectos relacionados à DA: (1) a identificação de CNVs que podem estar contribuindo para o desenvolvimento da forma multifatorial da DA, usando a técnica de array-CGH, e (2) a análise de alterações do padrão global de metilação do DNA no córtex frontal de indivíduos com a forma multifatorial da DA, usando um microarranjo que interroga o status de metilação de 450.000 sítios CpGs. Em nossa investigação sobre desequilíbrios genômicos na DA, identificamos 6 CNVs raras com conteúdo gênico relevante para o fenótipo investigado. Dois indivíduos distintos do grupo DA apresentam microduplicações em genes que codificam diferentes subunidades do mesmo tipo de canal de Ca2+ dependente de voltagem, o tipo L. Além disso, dos outros genes selecionados como especialmente interessantes, 4 estão envolvidos em diferentes processos inflamatórios e 1 é responsável por codificar a enzima nicotinamida fosforibosiltransferase, participante importante da via de biossíntese da molécula nicotinamida adenina dinucleotídeo (NAD). A implicação de um possível envolvimento de mediadores da sinalização celular do Ca2+ e da via de biossíntese da NAD na etiologia da DA também foi reforçada pelos nossos resultados sobre o padrão de metilação do DNA na DA. Dois genes importantes para a homeostasia intracelular do Ca2+ e via de biossíntese da NAD apresentaram sítios CpGs diferenciamente metilados nos sujeitos com DAAlzheimer\'s disease (AD) is the most common form of dementia in the population, corresponding to 50-60% of all cases. Although clinical diagnosis seems to be accurate, the definitive diagnosis of the disease can only be made by a post mortem neuropathological exam that certifies the presence of the two hallmarks of AD: the accumulation of extracellular senile plaques containing β-amyloid (Aβ) and the intracellular neurofibrillary tangles containing hyperphosphorylated tau protein. Four genes are known to be involved in the etiology of AD, three of them (APP, PSEN1 and PSEN2) are associated to the familial form of the disease, which show autosomal dominant inheritance and correspond to the more severe and rare type of AD. Despite many genome wide association studies (GWAS), APOE still remains the only unequivocal genetic risk factor associated to the multifactorial form of AD. The discoveries from GWAS using SNPs collectively explain only a small percentage of heritable variation that may contribute in AD risk. Currently, new approaches have been used to investigate the genetic basis of the phenotypical variability inheritance that can influence the susceptibility of complex diseases. The important role of DNA copy number variation (CNV) has been demonstrated by several studies over the last years and shows that genomic imbalances may also significantly contribute to resistance or susceptibility to various complex diseases. Additionally, there is now increasing interest in exploring how epigenetic modifications, in particular DNA methylation, could influence complex diseases etiology. Thus, the major aim of this work were to investigate two aspects related to the multifactorial form of AD: (1) identification of rare CNVs, using array-CGH, that could contribute to the development of the disease, and (2) analysis of the DNA methylation pattern in frontal cortex of individuals with AD. In our study, we identified 6 rare CNVs with relevant gene content to the investigated phenotype. Two distinct subjects with AD from our casuistic presented microduplications in genes that encode different subunits of the same type of Ca2+ voltage channel, the L-type. Furthermore, among the other selected genes, four are involved in different inflammatory process and one encodes the nicotinamide phosphoribosyltransferase enzyme, important mediator of nicotinamide adenine dinucleotide (NAD) biosynthesis. The implication of a possible involvement of Ca2+ intracellular signaling mediators and NAD biosynthesis pathway in the etiology of AD was also reinforced by our analysis of DNA methylation pattern. Interestingly, two important genes, one to intracellular Ca2+ homeostasis and the other to NAD biosynthesis pathway presented CpGs sites differently methylated in the AD subject

Glutamate modulation of melatonin synthesis in the rat pineal gland.

Esse trabalho teve como objetivo estudar os efeitos do glutamato sobre a síntese de melatonina, avaliando a participação dos diferentes tipos de receptores e uma possível interação entre pinealócitos e astrócitos; além de avaliar o papel do fator de transcrição NF-kB nos efeitos modulatórios do glutamato. Para tanto, glândulas pineais de ratos foram mantidas em cultura e estimuladas com diferentes concentrações de glutamato e também com diversos agonistas glutamatérgicos. Culturas de pinealócitos isolados e co-culturas de pinealócitos e astrócitos também foram usadas. A melatonina foi quantificada em HPLC com detecção eletroquímica. Para caracterização dos receptores de glutamato foi feita uma análise de RT-PCR, e a ativação do NF-kB foi avaliada por ensaio de gel de retardo. Os resultados mostram que o glutamato exerce um efeito inibitório sobre a síntese de melatonina através de uma ação sobre os receptores metabotrópicos dos grupos I e II e ionotrópico do tipo AMPA e que as ações do glutamato são mediadas pela ativação do NF-kB dos astrócitos.The aim of this work was to study the effects of glutamate on melatonin synthesis, investigating the glutamate receptors involved and a possible interaction between the two predominant cell types of the pineal gland, pinealocytes and astrocytes; moreover, it was investigated the involvement of the transcription factor NF-kB on the glutamate effects. To accomplish this, pineal glands were kept in culture and stimulated with glutamate or glutamate agonists. Isolated pinealocytes or in association with astrocytes in culture were also stimulated with glutamate. Melatonin was quantified by HPLC with electrochemical detection. Glutamate receptors were characterized by RT-PCR and NF-kB activation was evaluated by gel shift assay. The data showed that glutamate has an inhibitory effect on melatonin synthesis that is mediated by groups I and II glutamate metabotropic receptors and AMPA receptor and that this effect involves the activation of astrocytic NF-kB

Glutamate modulation of melatonin synthesis in the rat pineal gland.

Esse trabalho teve como objetivo estudar os efeitos do glutamato sobre a síntese de melatonina, avaliando a participação dos diferentes tipos de receptores e uma possível interação entre pinealócitos e astrócitos; além de avaliar o papel do fator de transcrição NF-kB nos efeitos modulatórios do glutamato. Para tanto, glândulas pineais de ratos foram mantidas em cultura e estimuladas com diferentes concentrações de glutamato e também com diversos agonistas glutamatérgicos. Culturas de pinealócitos isolados e co-culturas de pinealócitos e astrócitos também foram usadas. A melatonina foi quantificada em HPLC com detecção eletroquímica. Para caracterização dos receptores de glutamato foi feita uma análise de RT-PCR, e a ativação do NF-kB foi avaliada por ensaio de gel de retardo. Os resultados mostram que o glutamato exerce um efeito inibitório sobre a síntese de melatonina através de uma ação sobre os receptores metabotrópicos dos grupos I e II e ionotrópico do tipo AMPA e que as ações do glutamato são mediadas pela ativação do NF-kB dos astrócitos.The aim of this work was to study the effects of glutamate on melatonin synthesis, investigating the glutamate receptors involved and a possible interaction between the two predominant cell types of the pineal gland, pinealocytes and astrocytes; moreover, it was investigated the involvement of the transcription factor NF-kB on the glutamate effects. To accomplish this, pineal glands were kept in culture and stimulated with glutamate or glutamate agonists. Isolated pinealocytes or in association with astrocytes in culture were also stimulated with glutamate. Melatonin was quantified by HPLC with electrochemical detection. Glutamate receptors were characterized by RT-PCR and NF-kB activation was evaluated by gel shift assay. The data showed that glutamate has an inhibitory effect on melatonin synthesis that is mediated by groups I and II glutamate metabotropic receptors and AMPA receptor and that this effect involves the activation of astrocytic NF-kB

Do Copy Number Changes in CACNA2D2, CACNA2D3, and CACNA1D Constitute a Predisposing Risk Factor for Alzheimer's Disease?

Dysregulation of calcium (Ca(2+)) homeostasis is now being recognized to be a key step in the pathogenesis of Alzheimer's disease (AD). Data from the literature, in particular the association between AD and polymorphism that interfere with Ca(2+) homeostasis indicates the presence of genetic factors in this process; further, presenilins mutations, which are known to cause the familial form of AD, are involved in the regulation of intracellular Ca(2+) stores. Here, we wish to draw attention to rare DNA copy number variations identified in two subjects with late-onset AD that led to partial or full duplication of genes that encode different subunits of the same type of voltage-gated Ca(2+) channels; these duplications of voltage-gated Ca(2+) channel genes is consistent with the critical role of calcium signaling in molecular processes underlying memory as has been demonstrated by several studies