Histopathological Characterization and Whole Exome Sequencing of Ectopic Thyroid: Fetal Architecture in a Functional Ectopic Gland from Adult Patient

Ectopic thyroid results from a migration defect of the developing gland during embryogenesis causing congenital hypothyroidism. But it has also been detected in asymptomatic individuals. This study aimed to investigate the histopathological, functional, and genetic features of human ectopic thyroids. Six samples were histologically examined, and the expression of the specific thyroid proteins was assessed by immunohistochemistry. Two samples were submitted to whole exome sequencing. An oropharynx sample showed immature fetal architecture tissue with clusters or cords of oval thyrocytes and small folliclesone sample exhibited a normal thyroid pattern while four showed colloid goiter. All ectopic thyroids expressed the specific thyroid genes and T4 at similar locations to those observed in normal thyroid. No somatic mutations associated with ectopic thyroid were found. This is the first immature thyroid fetal tissue observed in an ectopic thyroid due to the arrest of structural differentiation early in the colloid stage of development that proved able to synthesize thyroid hormone but not to respond to TSH. Despite the ability of all ectopic thyroids to synthetize specific thyroid proteins and T4, at some point in life, it may be insufficient to support body growth leading to hypothyroidism, as observed in some of the patients.FAPESP Grant [2009/53840-0]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), Sao Paulo, Brazil [2010/12005-9, 2014/24549-4]Instituto da TiroideUniv Sao Paulo FMUSP, Fac Med, Cellular & Mol Endocrine Lab, Thyroid Unit,LIM 25, Ave Doutor Arnaldo 455, BR-01246904 Sao Paulo, SP, BrazilSao Paulo Publ Hlth Serv, Adolfo Lutz Inst, Av Dr Arnaldo 355, BR-01246000 Sao Paulo, SP, BrazilHead & Neck Surg Santa Catarina Hosp, Av Paulista 200, BR-01310000 Sao Paulo, SP, BrazilUNESP, Botucatu Sch Med, Dept Internal Med, Av Prof Montenegro,S-N Dist Rubiao Jr, BR-18618687 Botucatu, SP, BrazilHosp Pediat Dr Juan Garrahan, Serv Endocrinol, Combate Pozos 1881,C1245AAM, Buenos Aires, DF, ArgentinaUniv Estadual Campinas, Fac Ciencias Med, Dept Cirurgia, Disciplina Cirurgia Cabeca & Pescoco, R Tessalia Vieira Camargo 126, BR-13083887 Campinas, SP, BrazilUniv Fortaleza Unifor, Med Sch, Av Washington Soares 1321, BR-60811905 Fortaleza, CE, BrazilUniv Fed Sao Paulo UNIFESP, Postgrad Program Biotechnol, Pedro Toledo 669, BR-04039903 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Postgrad Programs Biotechnol & Struct & Funct Bio, Dept Ciencias Biol, Thyroid Mol Sci Lab,UNIFESP, Pedro Toledo 669, BR-04039903 Sao Paulo, SP, BrazilHosp Sirio Libanes, Mol Oncol Ctr, Rua Prof Daher Cutait 69, BR-01308060 Sao Paulo, SP, BrazilUniv Fed Sao Paulo UNIFESP, Postgrad Program Biotechnol, Pedro Toledo 669, BR-04039903 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Postgrad Programs Biotechnol & Struct & Funct Bio, Dept Ciencias Biol, Thyroid Mol Sci Lab,UNIFESP, Pedro Toledo 669, BR-04039903 Sao Paulo, SP, BrazilFAPESP [2009/53840-0]FAPESP[2010/12005-9, 2014/24549-4]Web of Scienc

Use of computational methods to study the transcription and gene regulation in Homo sapiens and Mus musculus

O gene, uma seqüência de nucleotídeos necessária para a síntese de moléculas funcionais, é transcrito e regulado por um conjunto de processos e fatores extremamente complexos. Entender o momento e o tecido em que os genes são expressos, as isoformas funcionais, as regiões controladoras e os fatores envolvidos na regulação da expressão de cada gene é um dos grandes desafios da biologia molecular moderna. Hoje, com a enorme quantidade de informações de seqüências genômicas e de transcriptomas, aliado ao desenvolvimento de métodos computacionais para agrupar e analisar estes dados em larga escala, o estudo dos fenômenos relacionados à transcrição e regulação gênica está passando por uma revolução. Por exemplo, é possível medir, concomitantemente, a expressão gênica de milhares de genes em diferentes tecidos, assim como identificar diversos fenômenos que atuam nestes genes. Neste trabalho nós desenvolvemos e aplicamos métodos computacionais no estudo de quatro temas envolvendo aspectos chave da transcrição e regulação gênica. No primeiro trabalho, nós abordamos a expressão gênica tecido-específica através do estudo dos genes expressos no cérebro e em dez regiões cerebrais de camundongo. No segundo trabalho, nós identificamos seqüências potencialmente envolvidas no controle da transcrição gênica através do estudo de motivos sobre representados na região promotora dos genes de receptores olfativos. No terceiro trabalho, analisamos o transcriptoma humano quanto a presença de eventos de retenção de intron, um tipo de splicing alternativo. No quarto trabalho, nós abordamos a complexidade do transcriptoma e a regulação da expressão gênica através do estudo de pares de genes senso-antisenso em humanos e camundongos. Em todos os trabalhos, obtivemos resultados que nos permitiram tirar conclusões específicas sobre cada fenômeno estudado e nos mostraram a importância de estudá-los através de uma abordagem em larga escala. Adicionalmente, verificamos que os nossos métodos computacionais foram eficientes e adequados para o estudo da transcrição e regulação gênica em Homo sapiens e Mus musculus.Genes, nucleotide sequences necessary for the synthesis of functional molecules, are transcribed and regulated by extremely complex cellular and molecular processes. To understand when and in which tissues the genes are expressed, their functional isoforms, control regions and the factors involved in gene regulation is one of major challenges of modern molecular biology. Today, the availability of complete genome sequences and transcriptomes, together with the development of new computational methods allows the study of phenomena related to the transcription and gene regulation in a large scale. For example, it is possible to quantify, concomitantly, gene expression of thousands of genes in different tissues and analyze different aspects of their regulation. In this work we developed and applied computational methods to the study of four key aspects of gene transcription and regulation. In the first study, we addressed tissue specific gene expression through the study of genes that are preferentially expressed in the brain and ten different mouse brain regions. In the second study, we identified sequences that are potentially involved in the control of gene transcription through the study of motifs that are over represented in the promoter region of olfactory receptor genes. In the third study, we browsed the human for the presence of intron retention, a type of alternative splicing. In the fourth study, we addressed the transcriptoma complexity and gene expression regulation through the study of pair of sense-antisense genes in human and mouse. In all studies, our results allowed us to make specific conclusions about each phenomenon analyzed which showed us the importance of a large scale approach. In addition, we verified that our computational methods can be efficiently applied to the study of transcription and gene regulation in Homo sapiens and Mus musculus

Detection and evaluation of intron retention events in the human transcriptome

Alternative splicing is a very frequent phenomenon in the human transcriptome. There are four major types of alternative splicing: exon skipping, alternative 3′ splice site, alternative 5′ splice site, and intron retention. Here we present a large-scale analysis of intron retention in a set of 21,106 known human genes. We observed that 14.8% of these genes showed evidence of at least one intron retention event. Most of the events are located within the untranslated regions (UTRs) of human transcripts. For those retained introns interrupting the coding region, the GC content, codon usage, and the frequency of stop codons suggest that these sequences are under selection for coding potential. Furthermore, 26% of the introns within the coding region participate in the coding of a protein domain. A comparison with mouse shows that at least 22% of all informative examples of retained introns in human are also present in the mouse transcriptome. We discuss that the data we present suggest that a significant fraction of the observed events is not spurious and might reflect biological significance. The analyses also allowed us to generate a reliable set of intron retention events that can be used for the identification of splicing regulatory elements

A straightforward assay to evaluate DNA integrity and optimize next-generation sequencing for clinical diagnosis in oncology

Next generation sequencing (NGS) has become an informative tool to guide cancer treatment and conduce a personalized approach in oncology. The biopsy collected for pathologic analysis is usually stored as formalin fixed paraffin-embedded (FFPE) blocks and then availed for molecular diagnostic, resulting in DNA molecules that are invariably fragmented and chemically modified. In an attempt to improve NGS based diagnostics in oncology we developed a straightforward DNA integrity assessment assay based on qPCR, defining clear parameters to whether NGS sequencing results is accurate or when it should be analyzed with caution. We performed DNA extraction from 12 tumor samples from diverse tissues and accessed DNA integrity by straightforward qPCR assays. In order to perform a cancer panel NGS sequencing, DNA library preparation was performed using RNA capture baits. Reads were aligned to the reference human genome and mutation calls were further validated by Sanger sequencing. Results obtained by the DNA integrity assays correlated to the efficiency of the pre-capture library preparation in up to 0.94 (Pearson's test). Moreover, sequencing results showed that poor integrity DNA leads to high rates of false positive mutation calls, specially C:G > T:A and C:G > A:T. Poor quality FFPE DNA samples are prone to generating false positive mutation calls. These are especially perilous in cases in which subclonal populations are expected, such as in advance disease, since it could lead clinicians to erroneous conclusions and equivocated conduct

Evidence of Cooperation between Hippo Pathway and RAS Mutation in Thyroid Carcinomas

Thyroid cancer incidences have been steadily increasing worldwide and are projected to become the fourth leading cancer diagnosis by 2030. Improved diagnosis and prognosis predictions for this type of cancer depend on understanding its genetic bases and disease biology. RAS mutations have been found in a wide range of thyroid tumors, from benign to aggressive thyroid carcinomas. Based on that and in vivo studies, it has been suggested that RAS cooperates with other driver mutations to induce tumorigenesis. This study aims to identify genetic alterations or pathways that cooperate with the RAS mutation in the pathogenesis of thyroid cancer. From a cohort of 120 thyroid carcinomas, 11 RAS-mutated samples were identified. The samples were subjected to RNA-Sequencing analyses. The mutation analysis in our eleven RAS-positive cases uncovered that four genes that belong to the Hippo pathway were mutated. The gene expression analysis revealed that this pathway was dysregulated in the RAS-positive samples. We additionally explored the mutational status and expression profiling of 60 RAS-positive papillary thyroid carcinomas (PTC) from The Cancer Genome Atlas (TCGA) cohort. Altogether, the mutational landscape and pathway enrichment analysis (gene set enrichment analysis (GSEA) and Kyoto Encyclopedia of Genes and Genome (KEGG)) detected the Hippo pathway as dysregulated in RAS-positive thyroid carcinomas. Finally, we suggest a crosstalk between the Hippo and other signaling pathways, such as Wnt and BMP

Global changes in nitration levels and DNA binding profile of Trypanosoma cruzi histones induced by incubation with host extracellular matrix.

Adhesion of T. cruzi trypomastigotes to components of the extracellular matrix (ECM) is an important step in mammalian host cell invasion. We have recently described a significant increase in the tyrosine nitration levels of histones H2A and H4 when trypomastigotes are incubated with components of the ECM. In this work, we used chromatin immunoprecipitation (ChIP) with an anti-nitrotyrosine antibody followed by mass spectrometry to identify nitrated DNA binding proteins in T. cruzi and to detect alterations in nitration levels induced upon parasite incubation with the ECM. Histone H1, H2B, H2A and H3 were detected among the 9 most abundant nitrated DNA binding proteins using this proteomic approach. One nitrated tyrosine residue (Y29) was identified in Histone H2B in the MS/MS spectrum. In addition, we observed a significant increase in the nitration levels of histones H1, H2B, H2A and H4 upon parasite incubation with ECM. Finally, we used ChIP-Seq to map global changes in the DNA binding profile of nitrated proteins. We observed a significant change in the binding pattern of nitrated proteins to DNA after parasite incubation with ECM. This work provides the first global profile of nitrated DNA binding proteins in T. cruzi and additional evidence for modification in the nitration profile of histones upon parasite incubation with ECM. Our data also indicate that the parasite interaction with the ECM induces alterations in chromatin structure, possibly affecting nuclear functions

The Human Cell Surfaceome of Breast Tumors

Introduction. Cell surface proteins are ideal targets for cancer therapy and diagnosis. We have identified a set of more than 3700 genes that code for transmembrane proteins believed to be at human cell surface. Methods. We used a high-throuput qPCR system for the analysis of 573 cell surface protein-coding genes in 12 primary breast tumors, 8 breast cell lines, and 21 normal human tissues including breast. To better understand the role of these genes in breast tumors, we used a series of bioinformatics strategies to integrates different type, of the datasets, such as KEGG, protein-protein interaction databases, ONCOMINE, and data from, literature. Results. We found that at least 77 genes are overexpressed in breast primary tumors while at least 2 of them have also a restricted expression pattern in normal tissues. We found common signaling pathways that may be regulated in breast tumors through the overexpression of these cell surface protein-coding genes. Furthermore, a comparison was made between the genes found in this report and other genes associated with features clinically relevant for breast tumorigenesis. Conclusions. The expression profiling generated in this study, together with an integrative bioinformatics analysis, allowed us to identify putative targets for breast tumors

A straightforward assay to evaluate DNA integrity and optimize next-generation sequencing for clinical diagnosis in oncology

Next generation sequencing (NGS) has become an informative tool to guide cancer treatment and conduce a personalized approach in oncology. The biopsy collected for pathologic analysis is usually stored as formalin fixed paraffin-embedded (FFPE) blocks and then availed for molecular diagnostic, resulting in DNA molecules that are invariably fragmented and chemically modified. In an attempt to improve NGS based diagnostics in oncology we developed a straightforward DNA integrity assessment assay based on qPCR, defining clear parameters to whether NGS sequencing results is accurate or when it should be analyzed with caution. We performed DNA extraction from 12 tumor samples from diverse tissues and accessed DNA integrity by straightforward qPCR assays. In order to perform a cancer panel NGS sequencing, DNA library preparation was performed using RNA capture baits. Reads were aligned to the reference human genome and mutation calls were further validated by Sanger sequencing. Results obtained by the DNA integrity assays correlated to the efficiency of the pre-capture library preparation in up to 0.94 (Pearson's test). Moreover, sequencing results showed that poor integrity DNA leads to high rates of false positive mutation calls, specially C:G > T:A and C:G > A:T. Poor quality FFPE DNA samples are prone to generating false positive mutation calls. These are especially perilous in cases in which subclonal populations are expected, such as in advance disease, since it could lead clinicians to erroneous conclusions and equivocated conduct