Identification of a rare copy number polymorphic gain at 3q12.2 with candidate genes for familial endometriosis

Abstract Endometriosis is a complex disease that affects 10-15% of women of reproductive age. Familial studies show that relatives of affected patients have a higher risk of developing the disease, implicating a genetic role for this disorder. Little is known about the impact of germline genomic copy number variant (CNV) polymorphisms on the heredity of the disease. In this study, we describe a rare CNV identified in two sisters with familial endometriosis, which contain genes that may increase the susceptibility and progression of this disease. We investigated the presence of CNVs from the endometrium and blood of the sisters with endometriosis and normal endometrium of five women as controls without the disease using array-CGH through the Agilent 2x400K platform. We excluded common CNVs that were present in the database of genomic variation. We identified, in both sisters, a rare CNV gain affecting 113kb at band 3q12.2 involving two candidate genes: ADGRG7 and TFG. The CNV gain was validated by qPCR. ADGRG7 is located at 3q12.2 and encodes a G protein-coupled receptor influencing the NF-kappaβ pathway. TFG participates in chromosomal translocations associated with hematologic tumor and soft tissue sarcomas, and is also involved in the NF-kappa B pathway. The CNV gain in this family provides a new candidate genetic marker for future familial endometriosis studies. Additional longitudinal studies of affected families must confirm any associations between this rare CNV gain and genes involved in the NF-kappaβ pathway in predisposition to endometriosis.

Tracking of thermal microbial shock proteins and genomic variation of Bacillus cereus due to some abiotic stresses / Rastreamento de proteínas de choque microbiano térmico e variação genômica de Bacillus cereus devido a alguns estresses abióticos

Bacillus cereus is considered the most potent bacterial strain in terms of the increment in induced proteins during thermal treatment at 52 °C for 90 min. Protein production in food-born microorganism (Bacillus cereus) recovered from contaminated food was investigated in response to heat shock treatment. Bacterial tolerance towards pH, salinity, and temperature at various levels was also investigated. Heat-shock proteins (HSPs) produced when exposed to 52 °C for up to 60 minutes led to significant differences (30%) above the untreated control (37 °C), and the maximum difference was recorded at 52°C at 90 minutes. ISSR detected a higher number of bands/primer than RAPD (13.7 vs. 12.7, respectively), and more polymorphic bands (10.7 vs. 8.4 bands/primer, respectively). The untreated bacterial strain did not grow at pH levels lower than 3, whereas the thermally treated strain grew significantly at pH two. A consistent increase in HSPs was observed, with a gradual increase in salinity of less than 16%. Surprisingly, the gradual increase in temperature did not induce tolerance against higher temperatures. However, a significant growth rate was noticed in response to heat-shocked treatments. The untreated Bacillus cereus demonstrated antibiotic resistance to gentamycin and clindamycin (1.54 and 1.65 cm, respectively), much lower than the corresponding inhibition areas with preheat-treated test pathogen which were recorded (2.37 and 2.49 cm, respectively).

Bacillus cereus é considerada a cepa bacteriana mais potente em termos de incremento de proteínas induzidas durante o tratamento térmico a 52 °C por 90 min. A produção de proteínas em microorganismos de origem alimentar (Bacillus cereus) recuperados de alimentos contaminados foi investigada em resposta ao tratamento de choque térmico. A tolerância bacteriana ao pH, salinidade e temperatura em vários níveis também foram investigadas. Proteínas de choque térmico (HSPs) produzidas quando expostas a 52 °C por até 60 minutos levaram a diferenças significativas (30%) acima do controle não tratado (37 °C), e a diferença máxima foi registrada a 52 °C em 90 minutos . O ISSR detectou um maior número de bandas/iniciador do que o RAPD (13,7 vs. 12,7, respectivamente) e mais bandas polimórficas (10,7 vs. 8,4 bandas/iniciador, respectivamente). A cepa bacteriana não tratada não cresceu em níveis de pH abaixo de 3, enquanto a cepa tratada termicamente cresceu significativamente em pH dois. Observou-se aumento consistente de HSPs, com aumento gradual da salinidade inferior a 16%. Surpreendentemente, o aumento gradual da temperatura não induziu tolerância a temperaturas mais altas. No entanto, uma taxa de crescimento significativa foi observada em resposta aos tratamentos de choque térmico. O Bacillus cereus não tratado demonstrou resistência antibiótica à gentamicina e clindamicina (1,54 e 1,65 cm, respectivamente), muito menor do que as áreas de inibição correspondentes com patógeno de teste pré-tratado que foram registradas (2,37 e 2,49 cm, respectivamente).

High-Resolution Characterization of KIR Genes in a Large North American Cohort Reveals Novel Details of Structural and Sequence Diversity.

The KIR (killer-cell immunoglobulin-like receptor) region is characterized by structural variation and high sequence similarity among genes, imposing technical difficulties for analysis. We undertook the most comprehensive study to date of KIR genetic diversity in a large population sample, applying next-generation sequencing in 2,130 United States European-descendant individuals. Data were analyzed using our custom bioinformatics pipeline specifically designed to address technical obstacles in determining KIR genotypes. Precise gene copy number determination allowed us to identify a set of uncommon gene-content KIR haplotypes accounting for 5.2% of structural variation. In this cohort, KIR2DL4 is the framework gene that most varies in copy number (6.5% of all individuals). We identified phased high-resolution alleles in large multi-locus insertions and also likely founder haplotypes from which they were deleted. Additionally, we observed 250 alleles at 5-digit resolution, of which 90 have frequencies ≥1%. We found sequence patterns that were consistent with the presence of novel alleles in 398 (18.7%) individuals and contextualized multiple orphan dbSNPs within the KIR complex. We also identified a novel KIR2DL1 variant, Pro151Arg, and demonstrated by molecular dynamics that this substitution is predicted to affect interaction with HLA-C. No previous studies have fully explored the full range of structural and sequence variation of KIR as we present here. We demonstrate that pairing high-throughput sequencing with state-of-art computational tools in a large cohort permits exploration of all aspects of KIR variation including determination of population-level haplotype diversity, improving understanding of the KIR system, and providing an important reference for future studies.

In silico analysis of genomic variables associated to HPV16 integration sites / Análisis in silico de variables genómicas asociadas a sitios de integración de HPV16

Background: Despite current prophylactic interventions, a significant proportion of patients suffers a cancer-specific mortality, leading to a global awareness of the importance of identifying factors associated to the etiology of HPV-associated cancer. According to this, HPV-DNA integration into human genome is an important event in the pathogenesis. Purpose: To identify in silico, molecular regions of the genome where the HPV integration events occur Methods: We performed a bioinformatic study based on a systematic search in Medline through PubMed, Embase and Lilacs from inception to April 2019. We used the UCSC Genome Browser Home (https://genome.ucsc.edu) to evaluate the genetic environment. Results: HPV integration sites by anatomical location related to cervical cancer were 374 (61%). In addition, 325 (87%) of these integration sites had HPV-16, 21 (5%) had HPV-18 and 28 (7%) had another type of genotype. Oro-pharyngeal cavity was the second anatomic site with 162 (26%) integration sites. It is noteworthy that the HPV-16 was found integrated into 160 (99%) analyzed sites. Conclusion: Our results suggest that many of the integration sites reported in the scientific literature are HPV 16 from squamous cell carcinomas and 50% of HPV16 were integrated into transcriptional units that might affect the expression of gene target.

Antecedentes: A pesar de las intervenciones profilácticas actuales, una proporción significativa de pacientes muere debido al cáncer, lo que aumenta la conciencia global de la importancia de identificar los factores asociados a la etiología del cáncer asociado al VPH. Según esto, la integración del ADN-VPH en el genoma humano es un evento importante en la patogénesis. Propósito: Identificar in silico, las regiones moleculares del genoma donde ocurren los eventos de integración del VPH Métodos: Realizamos un estudio bioinformático basado en una búsqueda sistemática en Medline a través de PubMed, Embase y Lilacs desde el inicio hasta abril de 2019. Utilizamos el UCSC Genome Browser Home (https://genome.ucsc.edu) para evaluar el entorno genético. Resultados: Los sitios de integración del VPH relacionados con el cáncer de cuello uterino fueron 374 (61%). Además, 325 (87%) de estos sitios de integración tenían VPH-16, 21 (5%) tenían VPH-18 y 28 (7%) tenían otro tipo de genotipo. La cavidad orofaríngea fue el segundo sitio anatómico con 162 (26%) sitios de integración. Es de destacar que el VPH-16 se encontró integrado en 160 (99%) sitios analizados. Conclusión: Nuestros resultados sugieren que muchos de los sitios de integración reportados en la literatura científica que presentan al VPH-16 son carcinomas de células escamosas y que el 50% de estos VPH-16 se integraron en unidades transcripcionales que podrían afectar la expresión de algún gen objetivo.

Intraspecific Genomic Divergence and Minor Structural Variations in Leishmania (Viannia) panamensis.

Leishmania (Viannia) panamensis is one of the most important Leishmania species associated with cutaneous leishmaniasis (CL) in Latin America. Despite its wide geographic distribution and pathogenic potential in humans and animals, the genomic variability of this species is low compared with other Leishmania species circulating in the same geographical area. No studies have reported a detailed analysis of the whole genome of L. panamensis from clinical isolates using DNA high-throughput sequencing to clarify its intraspecific genomic variability or plausible divergence. Therefore, this study aimed to evaluate the intraspecific genomic variability of L. panamensis from Colombia and Panama. A total of 22 genomes were analyzed, 19 from Colombian patients with CL and three genomes from Panama obtained from public databases. The phylogenomic analysis revealed the potential existence of three well-supported clades as evidence of intraspecific divergence. Additionally, the whole-genome analysis showed low structural variations in terms of ploidy, copy number variations, and single-nucleotide polymorphisms (SNPs). SNPs shared among all clades were identified, revealing their importance in different biological processes of L. panamensis. The findings not only expand our knowledge of intraspecific genomic variability of one of the most important Leishmania species in South America but also highlights the possible existence of different clades/lineages/subpopulations across a geographic scale.

Phenotype comparison among individuals with developmental delay/intellectual disability with or without genomic imbalances.

BACKGROUND: The chromosomal microarray analysis (CMA) is recommended as a first-tier test for individuals with developmental delay (DD)/intellectual disability (ID) and/or multiple congenital anomalies. However, owing to high costs, this technique is not widely performed for diagnostic purposes in several countries. The aim of this study was to identify clinical features that could favour the hypothesis of genomic imbalances (GIs) in individuals with DD/ID. METHODS: The sample consisted of 63 individuals, and all of them underwent a detailed evaluation by a clinical geneticist and were investigated by the CMA. They were divided into two groups. Group A composed of 20 individuals with pathogenic copy number variants (CNVs); and group B composed of 43 individuals with normal CMA results or variants of uncertain clinical significance (VUS). RESULTS: Pathogenic GIs were found in 20 cases (32%), including 11 individuals with an abnormal karyotype, VUS was found in five individuals (8%) and the results were normal in 38 individuals (60%). Major anomalies were found in 15/20 (75%) individuals in group A against 35/43 (81%) in group B. Dysmorphisms (≥5) were found in 17/20 (85%) in group A and 41/43 (95%) in group B. The most frequent major anomalies detected in group A were congenital heart disease, epilepsy and renal malformation; and in group B, they were malformations of central nervous system, congenital heart disease, microcephaly, epilepsy and hearing impairment. There was no significant statistical difference among the frequencies in groups A and B. CONCLUSIONS: Evidences point that every individual with DD/ID, with no specific clinical suspicion, should have screening for GIs as a first-tier test, regardless of the presence or absence of additional major anomalies or dysmorphisms. Future studies with a similar design would be helpful, especially in countries where the access to new technologies is still limited.

Epidemiología molecular y diversidad viral de HBV y HDV / Molecular epidemiology and viral diversity of HBV and HDV

INTRODUCCIÓN La infección por el virus de la hepatitis B (VHB) es un problema de salud pública mundial. El virus de la hepatitis D (HDV) requiere la función auxiliar de HBV para su ensamblaje y transmisión. Argentina muestra una baja prevalencia de infecciones por VHB y VHD, pero esto puede variar según la región geográfica y el grupo vulnerable buscado. OBJETIVO Estudiar la epidemiología molecular del HBV y HDV en una población hospitalaria del conurbano bonaerense y observar marcadores de infección eventualmente asociados. METODOLOGÍA Se realizó un estudio molecular de 152 muestras de plasma con marcadores de infección por HBV. Para ello, se extrajo ADN y se amplificó la región parcial del S/P y preC/C de HBV por PCR y n-PCR, respectivamente. Para estudiar HDV en el total de muestras, se extrajo ARN y se amplificó la región parcial Delta por n-PCR además se realizó serológica para HDV por Elisa (anti-HDV; Abott). Las muestras positivas se secuenciaron y los árboles filogenéticos de distancia (N-J) se construyeron con el programa MEGA. Para analizar otros marcadores de infección asociados se utilizaron ELISAs para HCV y HIV (Abbott). RESULTADOS Del total muestras analizadas con HBsAg y/o anticuerpos anti-HBc reactivos, se detectaron 21 casos positivos para las regiones S/P y/o preC-C de HBV. A partir del análisis filogenético, se observó la circulación de los subgenotipos A1, A2, D3, F1b, F3 y F4. Para HDV, fueron 40 los casos positivos de los cuales 8 fueron secuenciados, clasificando todos ellos dentro del genotipo 1. La prevalencia para anti-HDV fue 1,97% y para marcadores virales asociados se observó un 11,84% para anti-HCV y un 7,23% para HIV. DISCUSIÓN Se registró la circulación de diferentes genotipos de HBV y HDV previamente descripto en nuestro país. Los datos de prevalencia de HCV, HDV y HIV en individuos con marcadores serológicos de HBV demuestran la importancia de realizar el monitoreo de estos virus en esta población

Strategies to avoid drowning in the deep sequencing data flood

The enormous technological progress in the field of functional genomics during the last 15 years had a significant impact on animal sciences. With the development of Next Generation Sequencing it became feasible to analyze genomes and transcriptomes within short time frames and affordable costs. One major challenge of this rapid development is to manage the data flood and to perform data analysis and integration in an optimal manner. This review provides some information about a typical analysis pipeline for RNASequencing (RNA-Seq) data and a strategy for the analysis of small RNA-Seq data derived from species with poor annotation for non-coding RNA genes. Furthermore, problems regarding gene annotation in livestock species and their possible implications for data analysis and interpretation are discussed. Despite of not yet solved problems and challenges with respect to data analysis and integration the approaches in the field of functional genome analysis opened up new ways to try to understand the complex trait fertility.

Strategies to avoid drowning in the deep sequencing data flood

The enormous technological progress in the field of functional genomics during the last 15 years had a significant impact on animal sciences. With the development of Next Generation Sequencing it became feasible to analyze genomes and transcriptomes within short time frames and affordable costs. One major challenge of this rapid development is to manage the data flood and to perform data analysis and integration in an optimal manner. This review provides some information about a typical analysis pipeline for RNASequencing (RNA-Seq) data and a strategy for the analysis of small RNA-Seq data derived from species with poor annotation for non-coding RNA genes. Furthermore, problems regarding gene annotation in livestock species and their possible implications for data analysis and interpretation are discussed. Despite of not yet solved problems and challenges with respect to data analysis and integration the approaches in the field of functional genome analysis opened up new ways to try to understand the complex trait fertility.(AU)

Variação genotípica de Leishmania (Viannia) braziliensis permitindo sua adaptação no Estado de São Paulo e outras regiões brasileiras / Leishmania (Viannia) braziliensis genotypic variation associated with its adaptation in São Paulo State and other Brazilian regions

A leishmaniose cutânea ou tegumentar é uma doença tropical negligenciada causada por protozoários de dois subgêneros (Leishmania e Viannia). Normalmente produz úlceras em diferentes partes do corpo como face, pernas, braços, e em grande número, causando sérios problemas psicológicos e sociais. Leishmania (Viannia) braziliensis apresenta considerável importância clínica e é considerada a principal espécie causadora da leishmaniose cutânea Americana (LCA) no Brasil. No Estado de São Paulo, LCA é uma doença reemergente provocando lesões tegumentares, que são associadas a metástases mucosas. Neste artigo de revisão discute-se a complexa interação entre a diversidade de espécies de Leishmania, vetores, reservatórios silvestres e urbanos em variados ambientes geográficos. Todos esses fatores aliados às formas de resposta do hospedeiro originam as distintas formas clínicas. Aborda-se também a complexidade de elementos que envolvem a transmissão da LCA, as diversas formas clínicas que a doença apresenta, bem como, as dificuldades em se estabelecer o diagnóstico preciso, a diversidade de perfis epidemiológicos, a distribuição dos vetores, dados ecopidemiológicos da doença, e os mecanismos de adaptação de L. (V.) braziliensis. Diferentes estudos que envolvem a epidemiologia molecular contribuíram para o entendimento da constante adaptação desse parasita a seus diversos hospedeiros. Este artigo de revisão foi elaborado com base em artigos científicos, livros e portais eletrônicos apontando ampla distribuição associada a tipos heterogêneos de transmissão. Diferentes estudos atribuem a variabilidade genética exibida por L. (V.) braziliensis como fenômeno responsável pela adaptação em infectar múltiplos hospedeiros e vetores, assim como habitar em diferentes regiões geográficas...

Allopatric chromosomal variation in Nematocharax venustus Weitzman, Menezes & Britski, 1986 (Actinopterygii: Characiformes) based on mapping of repetitive sequences

Characiformes is the most cytogenetically studied group of freshwater Actinopterygii, but karyotypical data of several taxa remain unknown. This is the case of Nematocharax , regarded as a monotypic genus and characterized by marked sexual dimorphism. Therefore, we provide the first cytogenetic report of allopatric populations of Nematocharax venustus based on distinct methods of chromosomal banding and fluorescence in situ hybridization (FISH) with repetitive DNA probes (18S and 5S rDNA). The karyotype macrostructure was conserved in all specimens and populations, independently on sex, since they shared a diploid number (2n) of 50 chromosomes divided into 8m+26sm+14st+2a. The heterochromatin was mainly distributed at pericentromeric regions and base-specific fluorochrome staining revealed a single pair bearing GC-rich sites, coincident with nucleolar organizer regions (NORs). On the other hand, interpopulation variation in both number and position of repetitive sequences was observed, particularly in relation to 5S rDNA. Apparently, the short life cycles and restricted dispersal of small characins, such as N. venustus , might have favored the divergence of repetitive DNA among populations, indicating that this species might encompass populations with distinct evolutionary histories, which has important implications for conservation measures.

Characiformes é o grupo de Actinopterygii de água doce mais estudado citogeneticamente, porém dados cariotípicos de vários taxa permanecem desconhecidos. Este é o caso de Nematocharax , considerado um gênero monotípico e caracterizado pelo acentuado dimorfismo sexual. Em vista disso, nós fornecemos a primeira descrição citogenética de populações alopátricas de Nematocharax venustus , baseada em métodos distintos de bandamento cromossômico e hibridação fluorescente in situ (FISH) com sondas de DNA repetitivo (DNAr 18S e 5S). A macroestrutura cariotípica mostrou-se conservada em todos os espécimes e populações, independentemente do sexo, uma vez que compartilharam um número diploide (2n) de 50 cromossomos dividido em 8m+26sm+14st+2a. A heterocromatina distribuiu-se principalmente nas regiões pericentroméricas e a coloração com fluorocromos base-específicos revelou um único par portador de sítios GC-ricos, coincidentes com as regiões organizadoras de nucléolo (RONs). Por outro lado, foi observada uma variação interpopulacional no número e na posição das sequências repetitivas, especialmente em relação ao DNAr 5S. Aparentemente, ciclos de vida curtos e dispersão restrita dos pequenos caracídeos, tal como N. venustus , podem ter favorecido a divergência do DNA repetitivo entre as populações, indicando que essa espécie pode englobar populações com distintas histórias evolutivas, o que tem implicações importantes para medidas de conservação.

Allopatric chromosomal variation in Nematocharax venustus Weitzman, Menezes & Britski, 1986 (Actinopterygii: Characiformes) based on mapping of repetitive sequences

Characiformes is the most cytogenetically studied group of freshwater Actinopterygii, but karyotypical data of several taxa remain unknown. This is the case of Nematocharax , regarded as a monotypic genus and characterized by marked sexual dimorphism. Therefore, we provide the first cytogenetic report of allopatric populations of Nematocharax venustus based on distinct methods of chromosomal banding and fluorescence in situ hybridization (FISH) with repetitive DNA probes (18S and 5S rDNA). The karyotype macrostructure was conserved in all specimens and populations, independently on sex, since they shared a diploid number (2n) of 50 chromosomes divided into 8m+26sm+14st+2a. The heterochromatin was mainly distributed at pericentromeric regions and base-specific fluorochrome staining revealed a single pair bearing GC-rich sites, coincident with nucleolar organizer regions (NORs). On the other hand, interpopulation variation in both number and position of repetitive sequences was observed, particularly in relation to 5S rDNA. Apparently, the short life cycles and restricted dispersal of small characins, such as N. venustus , might have favored the divergence of repetitive DNA among populations, indicating that this species might encompass populations with distinct evolutionary histories, which has important implications for conservation measures.(AU)

Characiformes é o grupo de Actinopterygii de água doce mais estudado citogeneticamente, porém dados cariotípicos de vários taxa permanecem desconhecidos. Este é o caso de Nematocharax , considerado um gênero monotípico e caracterizado pelo acentuado dimorfismo sexual. Em vista disso, nós fornecemos a primeira descrição citogenética de populações alopátricas de Nematocharax venustus , baseada em métodos distintos de bandamento cromossômico e hibridação fluorescente in situ (FISH) com sondas de DNA repetitivo (DNAr 18S e 5S). A macroestrutura cariotípica mostrou-se conservada em todos os espécimes e populações, independentemente do sexo, uma vez que compartilharam um número diploide (2n) de 50 cromossomos dividido em 8m+26sm+14st+2a. A heterocromatina distribuiu-se principalmente nas regiões pericentroméricas e a coloração com fluorocromos base-específicos revelou um único par portador de sítios GC-ricos, coincidentes com as regiões organizadoras de nucléolo (RONs). Por outro lado, foi observada uma variação interpopulacional no número e na posição das sequências repetitivas, especialmente em relação ao DNAr 5S. Aparentemente, ciclos de vida curtos e dispersão restrita dos pequenos caracídeos, tal como N. venustus , podem ter favorecido a divergência do DNA repetitivo entre as populações, indicando que essa espécie pode englobar populações com distintas histórias evolutivas, o que tem implicações importantes para medidas de conservação.(AU)

Estudo funcional da região não estruturada de ligação entre os domínios BRCT em tandem da proteína BRCA1 / [Estudo funcional da região não estruturada de ligação entre os domínios BRCT em tandem da proteína BRCA1]

As vias de reparo ao dano de DNA (RDD) são essenciais para a manutenção da integridade genômica. Mutações em genes que atuam nessas vias podem contribuir para o desenvolvimento de câncer. BRCA1 é um gene extremamente polimórfico, codificando uma proteína envolvida em importantes eventos de sinalização nuclear em resposta ao dano de DNA. Portadores de mutações em BRCA1 que levam a uma proteína disfuncional podem ter o risco aumentado de desenvolvimento de câncer de mama e ovário em até 80%. Alterações em BRCA1, tais como inserções, deleções e mutações nonsense, podem ser inferidas como patogênicas, uma vez que a perda dos últimos 11 resíduos de aminoácidos na região C-terminal resulta em uma proteína não funcional. Mutações do tipo missense, assim como inserções e deleções que mantém o quadro de leitura, apresentam dificuldades na classificação. Estudos funcionais representam uma importante ferramenta para a classificação da patogenicidade dos variantes com baixa frequência na população (ou ainda não identificados). Em 2007, nosso grupo estabeleceu um ensaio funcional que correlaciona a atividade transcricional de BRCA1 e a integridade da região C-terminal da proteína (ensaio de TA) ­ essa região (aminoácidos 1396 a 1863) é capaz de recrutar a holoenzima RNA polimerase II. Assim, é possível correlacionar a atividade de um sistema repórter com a patogenicidade de uma mutação específica. Este estudo tem foco na região de ligação entre os domínios tBRCT, denominada linker (aminoácidos 1737 a 1759). Foram preditas todas as mutações naturais missense no linker (132 variantes) e, utilizando estratégias de bioinformática (Align-GVGD, SFIT e PolyPhen), foram estabelecidas os níveis de associação ao câncer. Variantes previamente estudados/classificados em outros trabalhos (16) não foram incluídos, resultando em um conjunto de 116 variantes. Somente 23 variantes foram preditos como associados ao câncer por todos algoritmos. Os variantes selecionados para o estudo foram gerados e clonados no vetor pCDNA3, codificando uma proteína de fusão (domínio de ligação ao DNA de GAL4 fusionado a BRCA1 C-terminal). As construções foram utilizadas para obtenção de dados funcionais através do ensaio de TA. Os experimentos foram originalmente conduzidos a 37oC. A maioria dos variantes (20) apresentou um perfil patogênico, enquanto 3 varinate, todos na posição 1740, apresentaram perfil semelhante aos controles não patogênicos. Esses variantes demonstraram uma variação significativa na atividade transcricional (tanto entre replicatas, quanto experimentos independentes), sugerindo um comportamento termossensível. Assim, todos os variantes foram testados a 30ºC. Os variantes na posição V1740 mantiveram um comportamento não patogênico, no entanto 9 variantes apresentaram aumento estatisticamente significativo na atividade transcricional. Não foram observadas alterações significativas na estrutura secundária do linker como consequência de substituições de aminoácidos quando analisados através do diagrama de Ramachandran. As diferenças observadas a 37ºC e 30ºC sugerem que o linker é, possivelmente, um segmento termossensível na estrutura de BRCA1. Todos os dados gerados serão avaliados em modelo matemático (VarCall) para determinar a patogenicidade dos variantes de BRCA1 abordados nesse estudo.

Snakebites as a largely neglected problem in the Brazilian Amazon: highlights of the epidemiological trends in the State of Amazonas

Envenoming snakebites are thought to be a particularly important threat to public health worldwide, especially in rural areas of tropical and subtropical countries. The true magnitude of the public health threat posed by snakebites is unknown, making it difficult for public health officials to optimize prevention and treatment. The objective of this work was to conduct a systematic review of the literature to gather data on snakebite epidemiology in the Amazon region and describe a case series of snakebites from epidemiological surveillance in the State of Amazonas (1974-2012). Only 11 articles regarding snakebites were found. In the State of Amazonas, information regarding incidents involving snakes is scarce. Historical trends show an increasing number of cases after the second half of the 1980s. Snakebites predominated among adults (20-39 years old; 38%), in the male gender (78.9%) and in those living in rural areas (85.6%). The predominant snake envenomation type was bothropic. The incidence reported by the epidemiological surveillance in the State of Amazonas, reaching up to 200 cases/100,000 inhabitants in some areas, is among the highest annual snakebite incidence rates of any region in the world. The majority of the cases were reported in the rainy season with a case-fatality rate of 0.6%. Snakebite envenomation is a great disease burden in the State of Amazonas, representing a challenge for future investigations, including approaches to estimating incidence under-notification and case-fatality rates as well as the factors related to severity and disabilities.

Amino Acid metabolism conflicts with protein diversity.

The 20 protein-coding amino acids are found in proteomes with different relative abundances. The most abundant amino acid, leucine, is nearly an order of magnitude more prevalent than the least abundant amino acid, cysteine. Amino acid metabolic costs differ similarly, constraining their incorporation into proteins. On the other hand, a diverse set of protein sequences is necessary to build functional proteomes. Here, we present a simple model for a cost-diversity trade-off postulating that natural proteomes minimize amino acid metabolic flux while maximizing sequence entropy. The model explains the relative abundances of amino acids across a diverse set of proteomes. We found that the data are remarkably well explained when the cost function accounts for amino acid chemical decay. More than 100 organisms reach comparable solutions to the trade-off by different combinations of proteome cost and sequence diversity. Quantifying the interplay between proteome size and entropy shows that proteomes can get optimally large and diverse.

HIV-1 tropism dynamics and phylogenetic analysis from longitudinal ultra-deep sequencing data of CCR5- and CXCR4-using variants.

OBJECTIVE: Coreceptor switch from CCR5 to CXCR4 is associated with HIV disease progression. The molecular and evolutionary mechanisms underlying the CCR5 to CXCR4 switch are the focus of intense recent research. We studied the HIV-1 tropism dynamics in relation to coreceptor usage, the nature of quasispecies from ultra deep sequencing (UDPS) data and their phylogenetic relationships. METHODS: Here, we characterized C2-V3-C3 sequences of HIV obtained from 19 patients followed up for 54 to 114 months using UDPS, with further genotyping and phylogenetic analysis for coreceptor usage. HIV quasispecies diversity and variability as well as HIV plasma viral load were measured longitudinally and their relationship with the HIV coreceptor usage was analyzed. The longitudinal UDPS data were submitted to phylogenetic analysis and sampling times and coreceptor usage were mapped onto the trees obtained. RESULTS: Although a temporal viral genetic structuring was evident, the persistence of several viral lineages evolving independently along the infection was statistically supported, indicating a complex scenario for the evolution of viral quasispecies. HIV X4-using variants were present in most of our patients, exhibiting a dissimilar inter- and intra-patient predominance as the component of quasispecies even on antiretroviral therapy. The viral populations from some of the patients studied displayed evidences of the evolution of X4 variants through fitness valleys, whereas for other patients the data favored a gradual mode of emergence. CONCLUSIONS: CXCR4 usage can emerge independently, in multiple lineages, along the course of HIV infection. The mode of emergence, i.e. gradual or through fitness valleys seems to depend on both virus and patient factors. Furthermore, our analyses suggest that, besides becoming dominant after population-level switches, minor proportions of X4 viruses might exist along the infection, perhaps even at early stages of it. The fate of these minor variants might depend on both viral and host factors.

Estudo de variações genômicas para a identificação de biomarcadores personalizados e novos alvos terapêuticos em tumores colorretais / Study of genomic variation to identify biomarkers and novel therapeutic targets in colorectal tumors

O câncer colorretal é um dos tipos de tumores mais frequentes no mundo. A atual dificuldade na avaliação correta da resposta ao tratamento torna necessário o desenvolvimento de novas abordagens de detecção tumoral. Atualmente, o sequenciamento genômico em larga escala permite um estudo mais compreensivo das alterações estruturais e de sequência presentes no tumor. A aplicação destas abordagens de maneira personalizada permite o desenvolvimento de biomarcadores tumor específicos que podem facilitar a avaliação de resposta ao tratamento e a presença de doença residual, bem como revelar alterações de sequência em genes capazes de servir de novos alvos terapêuticos. Neste estudo foi desenvolvida uma metodologia eficiente para a identificação de biomarcadores baseados na existência de variações estruturais em genomas de tumores de reto, eliminando a necessidade de sequenciamento do genoma normal do mesmo paciente e diminuindo portanto o custo da abordagem. Os biomarcadores encontrados para cada um dos seis pacientes foram utilizados para avaliar a presença de doença residual após o tratamento através da detecção de DNA tumoral circulante nas amostras de plasma coletadas em momentos diferentes do tratamento. O sequenciamento em baixa cobertura personalizado é portanto uma alternativa viável e promissora para avaliar a resposta ao tratamento em pacientes com tumores de reto. Na segunda parte do estudo, a análise de linhagens celulares de tumores colorretais revelou uma grande quantidade de mutações pontuais somáticas (SNVs e InDels) em genes codificadores para proteínas de superfície celular (surfaceoma). Estas alterações no surfaceoma indicam potenciais novos alvos para drogas e vias regulatórias alteradas neste tipo de tumor. Além disso, estas mutações pontuais também são responsáveis pela geração de epítopos com potencial imunogênico e estes novos epítopos podem ser aplicados como vacinas antitumorais personalizadas e já haviam sido propostos como uma alternativa terapêutica. A presença de novos epítopos, principalmente nas linhagens com elevadas taxas de mutação (resultante da instabilidade de microssatélites e mutações em genes de reparo de DNA tipo mismatch ou POLE), sugerem também um potencial uso de drogas moduladoras do sistema imune em pacientes com tumores que apresentam estas mesmas características. Portanto, o estudo de alterações genômicas em tumores primários e linhagens de câncer colorretal permitiu a detecção de variações estruturais que foram utilizadas como biomarcadores personalizados em pacientes com tumores de reto assim como a identificação de genes contendo mutações pontuais em linhagens celulares de câncer colorretal, que revelam potenciais novos alvos terapêuticos a serem explorados na clínica

Colorectal cancer is one of the more frequent tumor types in the world. To select the appropriate treatment course, it is necessary to develop more precise diagnostic approaches. The current availability of high throughput genome sequencing methods allows for a comprehensive characterization of the structural and sequence alterations present in each tumor. The use of tumor genome sequencing in a personalized setting can result in tumor specific biomarkers that help evaluate response to treatment and the presence of residual disease, improving the clinical management of these patients, and also reveal sequence alterations in genes capable of serving as new therapeutic targets. In this study we developed an efficient bioinformatics pipeline to identify biomarkers based on the existing structural alterations in rectal tumor genomes, eliminating the need to sequence the matched normal genome and therefore reducing the cost for this approach. The biomarkers found for each of the six patients were used to evaluate the presence of residual disease after treatment through the detection of circulating tumor DNA in plasma samples collected at different points during the treatment. Sequencing tumor genomes with low coverage is therefore a viable and promising alternative to follow up rectal cancer patient's response to treatment. In the second part of this study, the analysis of colorectal cancer cell lines revealed a large quantity of point mutations (SNVs and InDels) in genes coding for proteins located in the cell surface (surfaceome). These alterations in the surfaceome indicate potential new drug targets and altered pathways in this type of tumor. Furthermore, these point mutations are also responsible for the generation of new epitopes with immunogenic potential and these new epitopes can be applied as personalized tumor vaccines and had previously been proposed as a therapeutic alternative. The presence of new epitopes, especially in the cell lines with elevated mutation rates (resulting from MSI and mutations in DNA mismatch-repair genes or POLE), suggests a potential use of immune checkpoint target drugs in patients with tumors that share these genetic characteristics. With a large-scale bioinformatics approach, we detected new tumor epitopes resulting from point mutations, present in most of the cell lines used. The analysis of gene expression data puts into perspective both the somatic mutations found and which targets are promising as well as the development of therapies based on vaccines derived from tumor epitopes. In conclusion, the study of genomic alterations in primary tumors and colorectal cancer cell lines allowed the detection of structural variations that were used as personalized biomarkers in patients with rectal tumors as well as the identification of genes containing point mutations in colorectal cancer cell lines, that reveal potential new therapeutic targets to be explored in the clinical setting

A retrotransposição de mRNAs como fator de variabilidade genética no genoma humano e de outros primatas / The retrotransposition of mRNAs as a factor of genetic variability in the human and other primates genomes

Duplicação genica é uma das principais forças levando a evolução dos genomas eucarioto. O impacto de duplicações gênicas/genômicas vem sendo investigado a muito tempo em humanos e outros primatas. Um segundo mecanismo de duplicação gênica, a retrotransposição baseada em RNA maduros, vem sendo menos estudada devido ao seu potencial menor de gerar cópias funcionais. No entanto, recentemente, publicações descreveram retrocópias funcionais em humanos, roedores e mosca de fruta. Nesta tese, para investigar sobre retrocópias causando variabilidade genética no genoma de primatas, nós desenvolvemos a implementamos os métodos para detectar estas inserções. Utilizando nove genomas e transcriptomas publicamente disponíveis (sete primatas e dois roedores) nós confirmamos um número similar, porém, com origem independente, de retrocópias em primatas e roedores. Nós também encontramos um enriquecimento de retrocópias no genoma de Platyrrhini, possivelmente explicado pela expansão de L1PA7 e L1P3 nestes genomas. Posteriormente, nós analisamos a ortologia de retrocópias no genoma de primatas e encontramos 127 eventos específicos à linhagem humana. Nós também exploramos dados do projeto 1000 Genomes para detectar retrocópias polimórficas (retroCNVs germinativos) e encontramos 17 eventos, presentes no genoma referência humano, mas ausentes em mais de um indivíduo. Similarmente, nós investigamos novas retroduplicações de mRNAs no genoma humano, detectando 21 eventos ausentes do genoma referência. Finalmente, investigamos a existência de retroCNVs somáticos e descrevemos sete possíveis retrocópias somáticas. Apesar de sua possível insignificância, nós encontramos que algumas retrocópias compartilhadas entre todos os primatas, espécie específicas, e polimórficas podem ser expressas per se ou como transcritos quiméricos com genes hospedeiros. Sobretudo, nós encontramos que retrocópias são um fator importante da variabilidade genética inter-espécie, intra-espécie e intra-indivíduo e podem estar influenciando a evolução de mamíferos ao criar reservatórios de duplicações potencialmente funcionais

Gene duplication is a major driving force of evolution in eukaryotic genome. The impact of gene/genomic duplication has long been investigated in human and other primates. A second mechanism of gene duplication, retrotransposition, which is based on mature RNA, has been traditionally less studied due to their lower potential to generate functional copies. Recently, however, publications described functional retrocopies in humans, murines and drosophila. Here, to gain insights of the genetic variability arising from retrocopies on primate genomes, we developed and implemented the methods to detect these insertions. Using nine publicly available reference genomes and transcriptomes (seven primates and two rodents) we described a similar number independently arisen retrocopies in primates and rodents. We also found an enrichment of retrocopies in Platyrhinni genomes, putatively explained by the expansion of L1PA7 and L1P3 in these genomes. Next, we evaluated the orthology of retrocopies in primate genomes and found 127 events specific to human lineage. We also explored 1000 Genomes Project data to detect polymorphic events (germinative retroCNVs) on human populations and found 17 events, present on the reference genome, absent in more than one individual. Conversely, we also investigated new insertions of mRNA retroduplications in the human genome, detecting 21 events absent to the human reference genome. Finally, we evaluated the existence of somatic retroCNVs and described seven putative somatic retrocopies. Despite their putative insignificance, we found that some of these shared, specie-specific and polymorphic events may be expressed per se and as chimeric transcripts within host genes. Taken together, we found that retrocopies are a great factor of genetic variation interspecie, intraspecie e intraindividual and may be affecting mammal evolution by creating reservoirs of potentially functional duplications

Sequence variants in oxytocin pathway genes and preterm birth: a candidate gene association study.

BACKGROUND: Preterm birth (PTB) is a complex disorder associated with significant neonatal mortality and morbidity and long-term adverse health consequences. Multiple lines of evidence suggest that genetic factors play an important role in its etiology. This study was designed to identify genetic variation associated with PTB in oxytocin pathway genes whose role in parturition is well known. METHODS: To identify common genetic variants predisposing to PTB, we genotyped 16 single nucleotide polymorphisms (SNPs) in the oxytocin (OXT), oxytocin receptor (OXTR), and leucyl/cystinyl aminopeptidase (LNPEP) genes in 651 case infants from the U.S. and one or both of their parents. In addition, we examined the role of rare genetic variation in susceptibility to PTB by conducting direct sequence analysis of OXTR in 1394 cases and 1112 controls from the U.S., Argentina, Denmark, and Finland. This study was further extended to maternal triads (maternal grandparents-mother of a case infant, N=309). We also performed in vitro analysis of selected rare OXTR missense variants to evaluate their functional importance. RESULTS: Maternal genetic effect analysis of the SNP genotype data revealed four SNPs in LNPEP that show significant association with prematurity. In our case-control sequence analysis, we detected fourteen coding variants in exon 3 of OXTR, all but four of which were found in cases only. Of the fourteen variants, three were previously unreported novel rare variants. When the sequence data from the maternal triads were analyzed using the transmission disequilibrium test, two common missense SNPs (rs4686302 and rs237902) in OXTR showed suggestive association for three gestational age subgroups. In vitro functional assays showed a significant difference in ligand binding between wild-type and two mutant receptors. CONCLUSIONS: Our study suggests an association between maternal common polymorphisms in LNPEP and susceptibility to PTB. Maternal OXTR missense SNPs rs4686302 and rs237902 may have gestational age-dependent effects on prematurity. Most of the OXTR rare variants identified do not appear to significantly contribute to the risk of PTB, but those shown to affect receptor function in our in vitro study warrant further investigation. Future studies with larger sample sizes are needed to confirm the findings of this study.

Clinical findings in patients with GLI2 mutations--phenotypic variability.

Mutations in the human GLI2 gene were first reported in association with defective anterior pituitary formation, panhypopituitarism, and forebrain anomalies represented by typical holoprosencephaly (HPE) and holoprosencephaly-like (HPE-L) phenotypes and postaxial polydactyly. Subsequently, anophthalmia plus orbital anomalies, heminasal aplasia, branchial arch anomalies and polydactyly have also been incorporated into the general phenotype. Here we described six Brazilian patients with phenotypic manifestations that range from isolated cleft lip/palate with polydactyly, branchial arch anomalies to semi-lobar holoprosencephaly. Novel sequence variants were found in the GLI2 gene in patients with marked involvement of the temporomandibular joint (TMJ), a new clinical finding observed with mutations of this gene. Clinical, molecular and genetic aspects are discussed.