scholarly journals Genome-wide quantification of rare somatic mutations in normal human tissues using massively parallel sequencing

2016 ◽  
Vol 113 (35) ◽  
pp. 9846-9851 ◽  
Author(s):  
Margaret L. Hoang ◽  
Isaac Kinde ◽  
Cristian Tomasetti ◽  
K. Wyatt McMahon ◽  
Thomas A. Rosenquist ◽  
...  
Keyword(s):  
Massively Parallel Sequencing ◽  
Point Mutations ◽  
Genetic Alterations ◽  
Human Tissues ◽  
Molecular Barcoding ◽  
Normal Tissues ◽  
Rare Mutations ◽  
Normal Human ◽  
Nuclear Genomes ◽  
Mutation Spectra

We present the bottleneck sequencing system (BotSeqS), a next-generation sequencing method that simultaneously quantifies rare somatic point mutations across the mitochondrial and nuclear genomes. BotSeqS combines molecular barcoding with a simple dilution step immediately before library amplification. We use BotSeqS to show age- and tissue-dependent accumulations of rare mutations and demonstrate that somatic mutational burden in normal human tissues can vary by several orders of magnitude, depending on biologic and environmental factors. We further show major differences between the mutational patterns of the mitochondrial and nuclear genomes in normal tissues. Lastly, the mutation spectra of normal tissues were different from each other, but similar to those of the cancers that arose in them. This technology can provide insights into the number and nature of genetic alterations in normal tissues and can be used to address a variety of fundamental questions about the genomes of diseased tissues.

scholarly journals CLONAL GROWTH IN VITRO OF EPITHELIAL CELLS FROM NORMAL HUMAN TISSUES

1956 ◽  
Vol 104 (4) ◽  
pp. 615-628 ◽  
Author(s):  
Philip I. Marcus ◽  
Steven J. Cieciura ◽  
Theodore T. Puck
Keyword(s):  
Hela Cell ◽  
Single Cells ◽  
Cross Sectional Area ◽  
Plating Efficiency ◽  
Human Tissues ◽  
Sectional Area ◽  
Nutrient Media ◽  
Cross Sectional ◽  
Normal Tissues ◽  
Normal Human

Tissue culture strains of cells from four different normal human tissues—liver, conjunctiva, kidney, and appendix—have been grown by the plating procedure previously developed for the HeLa strain of cervical carcinoma cells. This technique results in colony formation from isolated single cells, in a manner completely analogous to the plating of bacteria in semisolid nutrient media. Clonal cell strains have been isolated from each cell type. All behaved exactly alike in all properties studied except that some differences in plating efficiency were displayed in some of the growth media employed. The cells from normal human tissues resembled the HeLa S3 carcinomatous cell in the following properties:— (a) Single cells displayed a plating efficiency close to 100 per cent in an appropriate medium. (b) They all grew as an epithelial sheet on glass, the cells being closely packed and polygonal in shape. (c) They had mean generation times of 20 to 23 hours in the nutrient media employed, (d) The mitotic frequency was constant, and therefore the duration of mitosis was the same for all the strains studied, (e) The incidence of multinuclearity and giant formation was very low and similar in both types of cells. (f) Both classes of cells had the same total volume, and the same nuclear cross-sectional area. (g) Both also showed a tendency to spread more in the presence of human serum (concentration of 20 per cent or more) than in porcine serum. However, this differential morphological response was much more marked in the HeLa cell than in those from normal tissues. The only difference noted in the behavior of these two groups of cells lay in the tendency of the cells from normal tissues always to exhibit a greater cross-sectional area when spread on glass than the HeLa cell in the same medium. The frequency of occurrence of different types of multinuclearity in the HeLa cell and cells from normal tissues has been measured. The data suggest that multinuclearity depends on two factors: a necessary, predisposing state in the cell, and a random, independent event causing the appearance of an additional nucleus in such a prepared cell.

scholarly journals Cancer Genetics and Epigenetics in Cancer Risk Assesment

2021 ◽  
Vol 5 (2) ◽  
pp. 41
Author(s):  
Anna Meiliana ◽  
Nurrani Mustika Dewi ◽  
Andi Wijaya
Keyword(s):  
Cancer Cells ◽  
Cancer Genetics ◽  
Point Mutations ◽  
Genetic Alterations ◽  
Genetic Mutations ◽  
Control Mechanisms ◽  
Normal Tissues ◽  
Knowing That ◽  
Whole Exome ◽  
Methylation Patterns

Compared to the normal tissues, cancer cells tend to have higher proliferation rate and often lost their ability to undergo apoptosis. In addition, cancer cells can separate themselves from their original tissue thus causing metastasis in other part of body. While undergoing program cell death, disordered cellular programming can happen. The main causes of this cellular programming anomaly are epigenetic and genetic alterations, which have been known as two separate mechanisms in carcinogenetic. A recent outcome of whole exome sequencing of thousands of human cancers has been the unexpected discovery of many inactivating mutations in genes that control the epigenome. These mutations have the potential to disturb the DNA methylation patterns, histone modifications, and nucleosome positioning, hence, the causing gene expression alternation. Genetic alteration of the epigenome therefore contributes to cancer just as epigenetic process can cause point mutations and disable DNA repair functions. Epigenetic mechanisms changes could cause genetic mutations, and genetic mutations in epigenetic regulators could cause epigenome changes. Knowing that epigenome play a major role in the hierarchy of gene control mechanisms suggests that mutations might have impact on multiple pathways related to cancer phenotype. This pinpoint the fact that recently, the way the genes are organized and controlled are suggested to be a relevant factor for human carcinogenesis.Keywords: cancer genetic, cancer epigenetic, oncogens, tumor suppressor genes, driver mutation, passenger mutation

scholarly journals Human Kallikrein 10 Expression in Normal Tissues by Immunohistochemistry

2002 ◽  
Vol 50 (9) ◽  
pp. 1247-1261 ◽  
Author(s):  
Constantina D. Petraki ◽  
Vassiliki N. Karavana ◽  
Liu-Ying Luo ◽  
Eleftherios P. Diamandis
Keyword(s):  
Leydig Cells ◽  
Biological Fluids ◽  
Human Tissues ◽  
Protein Levels ◽  
Normal Tissues ◽  
Tissue Extracts ◽  
Organ Specific ◽  
Normal Human ◽  
Neuroendocrine Organs ◽  
Mouse Antibody

The normal epithelial cell-specific 1 (NES1) gene (official name kallikrein gene 10, KLK10) was recently cloned and encodes for a putative secreted serine protease (human kallikrein 10, hK10). Several studies have confirmed that hK10 shares many similarities with the other kallikrein members at the DNA, mRNA, and protein levels. The enzyme was found in biological fluids, tissue extracts, and serum. Here we report the first detailed immunohistochemical (IHC) localization of hK10 in normal human tissues. We used the streptavidin-biotin method with two hK10-specific antibodies, a polyclonal rabbit and a monoclonal mouse antibody, developed in house. We analyzed 184 paraffin blocks from archival, current, and autopsy material, prepared from almost every normal human tissue. The staining pattern, the distribution of the immunostaining, and its intensity were studied in detail. Previously, we reported the expression of another novel human kallikrein, hK6, by using similar techniques. The IHC expression of hK10 was generally cytoplasmic and not organ-specific. A variety of normal human tissues expressed the protein. Glandular epithelia constituted the main immunoexpression sites, with representative organs being the breast, prostate, kidney, epididymis, endometrium, fallopian tubes, gastrointestinal tract, bronchus, salivary glands, bile ducts, and gallbladder. The choroid plexus epithelium, the peripheral nerves, and some neuroendocrine organs (including the islets of Langerhans, cells of the adenohypophysis, the adrenal medulla, and Leydig cells) expressed the protein strongly and diffusely. The spermatic epithelium of the testis expressed the protein moderately. A characteristic immunostaining was observed in Hassall's corpuscles of the thymus, oxyphilic cells of the thyroid and parathyroid glands, and chondrocytes. Comparing these results with those of hK6, we observed that both kallikreins had a similar IHC expression pattern.

scholarly journals Bisulfite-converted duplexes for the strand-specific detection and quantification of rare mutations

2017 ◽  
Vol 114 (18) ◽  
pp. 4733-4738 ◽  
Author(s):  
Austin K. Mattox ◽  
Yuxuan Wang ◽  
Simeon Springer ◽  
Joshua D. Cohen ◽  
Srinivasan Yegnasubramanian ◽  
...  
Keyword(s):  
Massively Parallel Sequencing ◽  
Massively Parallel ◽  
Clinical Samples ◽  
Low Frequencies ◽  
Parallel Sequencing ◽  
Molecular Barcoding ◽  
Bisulfite Treatment ◽  
Rare Mutations ◽  
Template Dna ◽  
Generation Sequencing

The identification of mutations that are present at low frequencies in clinical samples is an essential component of precision medicine. The development of molecular barcoding for next-generation sequencing has greatly enhanced the sensitivity of detecting such mutations by massively parallel sequencing. However, further improvements in specificity would be useful for a variety of applications. We herein describe a technology (BiSeqS) that can increase the specificity of sequencing by at least two orders of magnitude over and above that achieved with molecular barcoding and can be applied to any massively parallel sequencing instrument. BiSeqS employs bisulfite treatment to distinguish the two strands of molecularly barcoded DNA; its specificity arises from the requirement for the same mutation to be identified in both strands. Because no library preparation is required, the technology permits very efficient use of the template DNA as well as sequence reads, which are nearly all confined to the amplicons of interest. Such efficiency is critical for clinical samples, such as plasma, in which only tiny amounts of DNA are often available. We show here that BiSeqS can be applied to evaluate transversions, as well as small insertions or deletions, and can reliably detect one mutation among >10,000 wild-type molecules.

scholarly journals A body map of somatic mutagenesis in morphologically normal human tissues

2020 ◽  
Author(s):  
Ruoyan Li ◽  
Lin Di ◽  
Jie Li ◽  
Wenyi Fan ◽  
Yachen Liu ◽  
...  
Keyword(s):  
Somatic Mutations ◽  
Genomic Analysis ◽  
Driver Mutations ◽  
Human Tissues ◽  
Normal Tissues ◽  
Variable Extent ◽  
Normal Human ◽  
Body Map ◽  
Somatic Mutagenesis ◽  
Mutational Processes

AbstractSomatic mutations accumulated in normal tissues are associated with aging and disease. Here, we performed a comprehensive genomic analysis of 1,737 morphologically normal tissue biopsies (~ 600 cells each), mostly from the epithelia, of nine organs from five donors. We found that somatic mutation accumulations and clonal expansions are widespread, although with variable extent, in morphologically normal human tissues. Somatic copy number alterations were rarely detected, except for tissues from esophagus and cardia. Endogenous mutational processes like SBS1 and SBS5 are ubiquitous among normal tissues though exhibiting different relative activities. Exogenous mutational processes like SBS22 were found in different tissues from the same donor. We reconstructed the spatial somatic clonal architecture with sub-millimeter resolution. In epithelial tissues from esophagus and cardia, macroscopic somatic clones expanded to several millimeters were frequently seen, whereas in tissues from colon, rectum, and duodenum somatic clones were microscopic in size and evolved independently. Our study depicted a body map of somatic mutations and clonal expansions from the same individuals, and it revealed that the degree of somatic clonal expansion and enrichment of driver mutations are highly organ specific.

scholarly journals Expression of Transcription Factor CREM in Human Tissues

2021 ◽  
pp. 002215542110320
Author(s):  
Heidi Kaprio ◽  
Vanina D. Heuser ◽  
Katri Orte ◽  
Mikko Tukiainen ◽  
Ilmo Leivo ◽  
...  
Keyword(s):  
Transcription Factor ◽  
Dna Binding ◽  
Cancer Biology ◽  
Target Genes ◽  
Protein Isoforms ◽  
Human Tissues ◽  
Fusion Genes ◽  
Mesenchymal Tumors ◽  
Normal Tissues ◽  
Normal Human

Cyclic AMP element modulator (CREM) is a transcription factor best known for its intricate involvement in spermatogenesis. The CREM gene encodes for multiple protein isoforms, which can enhance or repress transcription of target genes. Recent studies have identified fusion genes, with CREM as a partner gene in many neoplastic diseases. EWSR1-CREM fusion genes have been found in several mesenchymal tumors and in salivary gland carcinoma. These genes encode fusion proteins that include the C-terminal DNA-binding domain of CREM. We used a transcriptomic approach and immunohistochemistry to study the expression of CREM isoforms that include DNA-binding domains across human tissues. We found that CREM protein is widely expressed in almost all normal human tissues. A transcriptomic analysis of normal tissues and cancer showed that transcription of CREM can be altered in tumors, suggesting that also wild-type CREM may be involved in cancer biology. The wide expression of CREM protein in normal human tissues and cancer may limit the utility of immunohistochemistry for identification of tumors with CREM fusions:

scholarly journals Human Kallikrein 13 Expression in Normal Tissues: An Immunohistochemical Study

2003 ◽  
Vol 51 (4) ◽  
pp. 493-501 ◽  
Author(s):  
Constantina D. Petraki ◽  
Vassiliki N. Karavana ◽  
Eleftherios P. Diamandis
Keyword(s):  
Nervous System ◽  
Human Tissue ◽  
Leydig Cells ◽  
Human Tissues ◽  
Primordial Follicles ◽  
Normal Tissues ◽  
Trophoblastic Cells ◽  
Endocrine Organs ◽  
The Central Nervous System ◽  
Normal Human

The human tissue kallikrein 13 gene (KLK13), encoding for hK13 protein, was recently cloned and characterized. Here we describe the immunohistochemical (IHC) localization of hK13 in normal human tissues and compare it with the expression of two other kallikreins, hK6 and hK10. We performed the streptavidin-biotin IHC method on 204 paraffin blocks from archival, current, and autopsy material prepared from almost every normal human tissue, using a polyclonal and a monoclonal hK13 antibody. The staining was cytoplasmic and both antibodies yielded similar results. The hK13 protein was revealed in a variety of tissues, mainly in glandular epithelia. Other epithelia that expressed hK13 included the urothelium, the spermatic epithelium, and the epithelium of the choroid plexus. hK13 was intensely immunoexpressed by some endocrine organs, such as the adenohypophysis, the thyroid gland, the parathyroid glands, the adrenal medulla, the Leydig cells of the testis, and the cells of the endocrine pancreas. Immunoreactivity was also observed in the primordial follicles, the corpus luteum, and sparse luteinized cells in the stroma of the ovary, the trophoblastic cells of the placenta, the Hassall's corpuscles of the thymus, and chondrocytes. Nerves and ganglia of the peripheral nervous system, and both neurons and glial cells in the central nervous system, were positive. In short, hK13 was expressed by many glandular epithelia, some endocrine organs, and some specialized epithelia and cells. Comparison of these data with hK6 and hK10 expression suggests that the three kallikreins have a similar IHC pattern in normal human tissues.

scholarly journals Evaluation of the prognostic value of CBXs in gastric cancer patients

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Mengya He ◽  
Limin Yue ◽  
Haiyan Wang ◽  
Feiyan Yu ◽  
Mingyang Yu ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Target Genes ◽  
Prognostic Significance ◽  
Disease Free Survival ◽  
Genetic Alterations ◽  
Genetic Mutation ◽  
The Cancer Genome Atlas ◽  
Normal Tissues ◽  
Interactive Analysis ◽  
Gastric Cancer Patients

AbstractChromobox (CBX) proteins were suggested to exert epigenetic regulatory and transcriptionally repressing effects on target genes and might play key roles in the carcinogenesis of a variety of carcinomas. Nevertheless, the functions and prognostic significance of CBXs in gastric cancer (GC) remain unclear. The current study investigated the roles of CBXs in the prognosis of GC using the Oncomine, The Gene Expression Profiling Interactive Analysis (GEPIA), UALCAN, The Cancer Genome Atlas (TCGA), and cBioPortal databases. CBX1/2/3/4/5 were significantly upregulated in GC tissues compared with normal tissues, and CBX7 was downregulated. Multivariate analysis showed that high mRNA expression levels of CBX3/8 were independent prognostic factors for prolonged OS in GC patients. In addition, the genetic mutation rate of CBXs was 37% in GC patients, and genetic alterations in CBXs showed no association with OS or disease-free survival (DFS) in GC patients. These results indicated that CBX3/8 can be prognostic biomarkers for the survival of GC patients.

scholarly journals Altered toxicity of the prion protein peptide PrP106–126 carrying the Ala117→Val mutation

2000 ◽  
Vol 346 (3) ◽  
pp. 785-791 ◽  
Author(s):  
David R. BROWN
Keyword(s):  
Point Mutation ◽  
Prion Protein ◽  
Prion Diseases ◽  
Point Mutations ◽  
Human Sequence ◽  
Gene Coding ◽  
Normal Human ◽  
Microtubule Formation ◽  
Β Sheet ◽  
Prion Protein Peptide

The inherited prion diseases such as Gerstmann-Sträussler-Scheinker syndrome (GSS) are linked to point mutations in the gene coding for the cellular isoform of the prion protein (PrPC). One particular point mutation A117V (Ala117 → Val) is linked to a variable pathology that usually includes deposition of neurofibrillary tangles. A prion protein peptide carrying this point mutation [PrP106-126(117V)] was generated and compared with a peptide based on the normal human sequence [PrP106-126(117A)]. The inclusion of this point mutation increased the toxicity of PrP106-126 which could be linked to an increased β-sheet content. An assay of microtubule formation in the presence of tau indicated that PrP106-126 decreased the rate of microtubule formation that could be related to the displacement of tau. PrP106-126 carrying the 117 mutation was more efficient at inhibiting microtubule formation. These results suggest a possible mechanism of toxicity for protein carrying this mutation via destabilization of the cytoskeleton and deposition of tau in filaments, as observed in GSS.

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