1 X-Genes


The rate of micronuclei counted on lymphocyte cultures from five healthy female donors, 27–80 years old, increased with age. Using pXBR1 probe, specific for the alphoid DNA of the X chromosome, the presence of this chromosome was investigated by FISH (fluoroscence in sity hybridization) in both micronucleic and metaphases. Both X aneuploidy and frequency of X chromosome per micronuclei increased with age. However, this overinvolvement of X chromosome was not sufficient to explain the overall increase of micronuclei with age, suggesting that autosomes are also involved. Thus, the higher increase of X than autosome aneyploidy in lymphocytes may result from both an excess of X choromosome losses and a better survival of cells with a monoosomy X.

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Current Opinion in Genetics & Development

Volume 7, Issue 2, April 1997, Pages 274-280

The (epi)genetic control of mammalian X-chromosome inactivation

Author links open overlay panelJeannie TLeebRudolfJaenischa

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In mammals, the X chromosome is uniquely capable of complete inactivation. Research in the past two years has validated the long-held hypothesis that the 'X-inactivation center' (Xic) controls events of X inactivation and that its resident gene Xist is not only required but is at least partially responsible for the cis-restriction of X inactivation. Progress has also been made in identifying genes within the Xic. Although Xist remains the only known required element, evidence now suggests that a separate element for X counting must exist and that the Xic may be entirely contained within a 450 kb sequence. This small region may be sufficient for both initiation and establishment of X inactivation.

X Chromosome Inactivation

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Long Non-Coding RNA


DNA Methylation




Gene Expression



X Chromosome

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Epigenetics of X Chromosome Inactivation

Tamar Dvash, Guoping Fan, in Handbook of Epigenetics, 2011

XCI Regulation During Development

XCI is a developmentally-regulated process that involves sequential acquisition of silencing markers on the X chromosome to be inactivated. Two different patterns of XCI exist: imprinted and random. The majority of XCI properties are shared between the two different patterns, yet some differences exist that reflect the nature and the degree of stability of inactivation. Most of the research concerning XCI in mammals has been conducted with the mouse model system. At the fertilization stage, the female mouse zygote has both X chromosomes active. The first inactivation during development occurs upon the first cleavage. This inactivation is imprinted and therefore only the paternal X chromosome is inactivated [4,5]. Later on, after the blastocyst has formed, cells from the inner cell mass (ICM) reactivate the inactive X [5,6]. At this stage the embryo has two types of XCI status; the ICM cells have both active X chromosomes while the trophectoderm and the primitive endoderm still retain their imprinted paternal XCI since the first cleavage. Then, only upon differentiation will the ICM cells again inactivate one of their X chromosomes but this time stochastically, in contrast to the first cleavage event [5,6]. Since the ICM cells are the origin of the embryo proper, the second round of inactivation will result in random XCI in each cell and throughout development its progenies will maintain that particular Xi. The primordial germ cells (PGC) are an exception in this regard since these cells again reactivate their Xi later on in mouse development (E11.5–E13.5) and this status is maintained in the female germ cells [7].

Both random and imprinted XCI are initiated by monoallelic Xist gene expression. This expression leads to a series of epigenetic modifications such as depletion of RNA polymerase II, transcription factors, and euchromatic markers (see Fig. 21.3). Imprinted XCI is temporary compared to the random XCI that remains stable from the moment of establishment throughout many cell divisions and across the entire lifespan. Therefore in order to establish stable random XCI, the mechanisms for CpG island methylation are employed [8]. This modification is considered to be more stable than histone modifications which are characteristic of imprinted XCI and early epigenetic events of random inactivation [9]. Although XCI occurs in a narrow time window during mouse development it is suggested that the kinetics of gene silencing varies. Existing evidence shows that genes located in the vicinity of the X chromosome inactivation center (XIC) are first silenced during differentiation [10].

Another interesting phenomenon in XCI is the "escape" from inactivation; although the majority of the genes on the Xi are subjected to complete silencing, some are able to express from both active and inactive X chromosomes. The exact mechanism for genes escaping XCI is not fully understood but a recent study using the transgene approach revealed that it is probably an intrinsic property of a specific locus. Random integration of BAC clones carrying normally silenced or escaped gene (Jarid1c) loci into the X chromosome of female ESC lines was able to recapitulate the endogenous expression pattern. The authors concluded that the DNA sequence itself is sufficient to determine whether a locus will be subjected to XCI [11].

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Epigenetic Mechanisms of Cellular Memory During Development

Nathan Montgomery, ... Scott Bultman, in Essentials of Stem Cell Biology (Second Edition), 2009

Reactivation and Imprinted XCI

Although XCI is stable in somatic cells, the Xi is reactivated in primordial germ cells during development of the germline. Following fertilization, XCI is re-established in the early mouse embryo within the first few cell divisions, but in an imprinted manner instead of stochastically. Xist is transcribed from the paternal X (Xp) chromosome, but not the maternal X (Xm) chromosome. Xist ncRNA spreads from the Xic along the rest of the Xp to inactivate it. At the blastocyst stage, imprinted XCI is maintained in the trophectoderm and primitive endoderm, which give rise to extra- embryonic tissues, such as the placenta. In contrast, imprinted XCI is not maintained in the inner cell mass (which ES cells are derived from), which give rise to all of the cells of the embryo proper (Plath et al., 2002). For reasons that are not yet clear, the Xp is reactivated and both X chromosomes are expressed in the epiblast until stochastic XCI occurs around the time of gastrulation. Interestingly, imprinted XCI might represent the ancestral form of XCI because it occurs in marsupials, but stochastic XCI does not.

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Cytoplasmic Inheritance Redux

Evan Charney, in Advances in Child Development and Behavior, 2013

4.5 X-chromosome Inactivation

X-chromosome inactivation (XCI) provides a good example as to how the various epigenetic mechanisms canvassed above can interact. X-chromosome activity changes dynamically in female offspring during PID due to a combination of epigenetic events including DNA methylation, histone modifications, and RNA-mediated silencing. In female embryos with two X chromosomes, one of the two X chromosomes is selected stochastically to be inactivated, a process known as XCI. XCI is triggered by an ncRNA, Xist, which coats the chromosome selected for silencing (Plath, Mlynarczyk-Evans, Nusinow, & Panning, 2002). This is followed by the recruitment of protein complexes involved in multiple epigenetic processes, distinct histone modifications such as H3 K4 demethylation, H3 K9 methylation, H4 deacetylation, and DNA hypermethylation of CpG dinucleotides along X-linked genes (Lee, 2003).

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Gametogenesis, Fertilization and Early Development

Kim L. McIntyre, ... Paul D. Waters, in Encyclopedia of Reproduction (Second Edition), 2018

X Chromosome Inactivation: Silencing One X in Females

X chromosome inactivation occurs in female therian mammals, where gene expression from one X chromosome is silenced in the somatic cells of females. The silent X chromosome condenses to form a distinct compact body (Barr body) within the nucleus. X chromosome inactivation is controlled by an elaborate molecular mechanism that is not fully understood, although seems to involve both structural and sequence elements of the X chromosome. In eutherians, a noncoding gene (called XIST) is expressed from the X chromosome chosen for silencing. The XIST RNA then coats the chromosome and recruits an array of protein complexes that mediate silencing. The mechanism underlying the accumulation and spreading of XIST RNA along the X chromosome is yet to be resolved, although long interspersed nuclear elements (LINE1s) on the X chromosome may be implicated. LINE1 element density on the human X chromosome is almost twofold that of the autosomes. LINE1 elements are particularly enriched around the XIST gene and in regions where silencing is most effective in human and several other eutherian species (excluding mouse). This is consistent with a role for LINE1 elements in localization of XIST RNA on the X chromosome in these species, although direct binding appears unlikely (reviewed in Gendrel and Heard, 2014).

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Pluripotent Stem Cells

Heiner Niemann, ... Joseph W. Carnwath, in Handbook of Stem Cells (Second Edition), 2013

X-Chromosome Inactivation after Somatic Cloning

X-chromosome inactivation is the developmentally regulated process by which one of the two X-chromosomes in female mammals is silenced early in development to provide dosage compensation for X-linked genes. A single X-chromosome is sufficient, as shown in XY males (Lyon, 1961). Although the mechanism of X-chromosome inactivation is not yet fully understood, the paternal X-chromosome is typically inactivated by DNA methylation and remains inactive in placental tissue, while in the embryo proper either the paternal or maternal X-chromosome can be randomly selected on a cell-by-cell basis for inactivation, leading to a mosaic pattern in adult cells (Hajkova and Surani, 2004). Findings in the mouse revealed that the paternal imprint in the inner cell mass (ICM), i.e., the pluripotent cells that give rise to the fetus, is erased from the paternal X-chromosome late in preimplantation development followed by random X-inactivation (Mak et al., 2004). The paternal X-chromosome is partly silent at fertilization and becomes fully inactivated at the two- or four-cell stage (Huynh and Lee, 2003; Okamoto et al., 2004). Female somatic nuclear transfer-derived embryos inherit one active and one inactive X-chromosome from the donor cell. Messenger RNA expression analysis of bovine embryos cloned from adult donor cells at the blastocyst stage revealed a significant upregulation of XIST (X-inactivating specific transcript) compared to in vitro- and in vivo-derived embryos. Expression of X-chromosome-related genes is delayed in cloned as compared to in vivo-derived embryos (Wrenzycki et al., 2002). Premature X-inactivation was observed for the X-chromosome linked inhibitor of apoptosis (XIAP) gene in in vitro-produced bovine embryos compared with their in vivo counterparts (Knijn et al., 2005). These findings indicate perturbation of X-chromosome inactivation has occurred by the blastocyst stage after somatic cloning or in vitro fertilization and culture. In female bovine cloned calves, aberrant expression patterns of X-linked genes and hypomethylation of XIST in various organs of stillborn calves were observed. Random inactivation of the X-chromosome was found in the placenta of deceased clones but skewed in that of live bovine clones (Xue et al., 2002). This aberrant expression pattern of X-chromosome inactivation initiated in the trophectoderm seems to have resulted from incomplete nuclear reprogramming. Similar findings were obtained in studies of cloned mouse embryos (Eggan et al., 2000).

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Homology Effects

Marjori A. Matzke, ... Antonius J.M. Matzke, in Advances in Genetics, 2002

C. X-chromosome inactivation and other types of dosage compensation

X-chromosome inactivation involves overlapping sense and antisense RNA (Xist and Tsix), which could form an RNA duplex (Lee, 2000). Inactivation spreads in cis along the X, probably by means of LINE-1 retroelements, which are particularly concentrated on the X chromosome (Bailey et al., 2000). Although the contributions of these noncoding RNAs and L1 repeats to X inactivation and methylation remain to be clarified (Chapter 2), it is intriguing that recurring players have been observed in epigenetic phenomena in plants and in mammals. To further solidify the connections between parasitic sequences, noncoding RNAs, and epigenetic phenomena, it has been suggested that the noncoding RNAs involved in X-chromosome dosage compensation in Drosophila are of TE or viral origin (Pannuti and Lucchesi, 2000).

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Neurobiology of Autism

Brent Wilkinson, Daniel B. Campbell, in International Review of Neurobiology, 2013

3 LncRNA in Fundamental Genetic Mechanisms

X-chromosome inactivation (XCI) is essential to the identity of female mammals and is a highly intricate process involving multiple lncRNAs that work together in order to ultimately downregulate mass quantities of genes for dosage compensation. X-inactive specific transcript (Xist) is a conserved lncRNA specifically expressed in the inactive X-chromosome and required for XCI (Brown et al., 1992; Brown, 1991). This was confirmed in XX murine embryonic stem cells (mESCs) by introducing a targeted deletion of Xist into a single allele. Following differentiation, the targeted X chromosome would always fail to inactivate and only the X chromosome expressing Xist would undergo XCI (Penny, Kay, Sheardown, Rastan, & Brockdorff, 1996). Xist acts by coating the chromosome to be inactivated and then recruiting polycomb-group proteins for inactivation via epigenetic mechanisms (Plath et al., 2003). By manipulating the expression of Xist, selective silencing of one copy of chromosome 21 was achieved in induced pluripotent stem cells (iPSCs) derived from patients with Down's syndrome. In the iPSC model, this corrected for the trisomy of chromosome 21 involved in the pathogenesis of Down's syndrome and improved deficiencies in proliferation and neural rosette formation (Jiang et al., 2013). In addition, it was recently discovered that there is also a human specific lncRNA, XACT, that coats the active X chromosome in pluripotent cells (Vallot et al., 2013). Xist displays a common trait among lncRNAs as they can associate with chromatin and recruit proteins with epigenetic functions in order to regulate the transcription of multiple genes.

LncRNAs have also been shown to be involved in genomic imprinting, the process by which genes are expressed in a parent-specific manner. The paternally expressed lncRNA, Antisense Igf2r (Air), silences three protein-coding genes (Igf2r, Slc22a2, and Slc22a3) located within the Igf2r cluster on the same allele (Sleutels, Zwart, & Barlow, 2002). Like Xist, Air is conserved between mice and humans (Yotova et al., 2008) and is involved in epigenetic regulation as it inactivates the Slc22a3 gene by recruiting G9a (a histone methyltransferase) (Nagano et al., 2008). Factors influencing genomic imprinting are of extreme importance as dysregulation of this process is hallmark of a number of conditions, including the neurodevelopmental disorders, Angelman Syndrome (AS), and Prader-Willi Syndrome (PWS) (Horsthemke & Wagstaff, 2008).

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Genotoxicities and infertility

Tirupapuliyur V. Damodaran, in Reproductive and Developmental Toxicology, 2011

Normal and abnormal X chromosome inactivation

X chromosome inactivation is most commonly studied in the context of female mammalian development, where it performs an essential role in dosage compensation. However, another form of X-inactivation takes place in the male, during spermatogenesis, as germ cells enter meiosis. This second form of X-inactivation, called meiotic sex chromosome inactivation (MSCI), has emerged as a novel paradigm for studying the epigenetic regulation of gene expression. New studies have revealed that MSCI is a special example of a more general mechanism called meiotic silencing of unsynapsed chromatin (MSUC), which silences chromosomes that fail to pair with their homologous partners and, in doing so, may protect against aneuploidy in subsequent generations. Furthermore, failure in MSCI is emerging as an important etiological factor in meiotic sterility (James and Turner, 2007). MSCI is believed to result from meiotic silencing of unpaired DNA because the X and Y chromosomes remain largely unpaired throughout first meiotic prophase. However, unlike X-chromosome inactivation in female embryonic cells, where 25–30% of X-linked structural genes have been reported to escape inactivation, X-linked mRNA-encoding genes during spermatogenesis have failed to reveal any X-linked gene that escapes the silencing effects of MSCI in primary spermatocytes; many X-linked miRNAs are transcribed and processed in pachytene spermatocytes. This unprecedented escape from MSCI by these X-linked miRNAs suggests that they may participate in a critical function at this stage of spermatogenesis, including the possibility that they contribute to the process of MSCI itself, and/or that they may be essential for post-transcriptional regulation of autosomal mRNAs during the late meiotic and early postmeiotic stages of spermatogenesis (Song et al., 2009). This also makes them a potential target for alteration by environmental factors.

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Y Chromosome






X Chromosome

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International Review of Cell and Molecular Biology

Eleanor M. Maine, in International Review of Cell and Molecular Biology, 2010

3.3 Genome-wide analysis of germ line gene expression

X chromosome gene expression in the C. elegans germ line has also been explored using gene expression profiling and functional genomic analysis (Maeda et al., 2001; Piano et al., 2000, 2002; Reinke et al., 2000, 2004). Gene expression profiling has identified large sets of transcripts that are enriched in the germ line relative to the soma; these genes can be grouped into several categories based on expression pattern, including (i) germ line-intrinsic genes expressed in the XX and XO germ line, (ii) spermatogenesis-specific genes expressed in the XO and larval XX germ lines, and (iii) oogenesis-specific genes expressed in the female and adult hermaphrodite germ line (Reinke et al., 2000, 2004). These studies revealed that germ line-intrinsic and spermatogenesis-specific genes are underrepresented on the X chromosome relative to autosomes. In contrast, oogenesis-specific genes are not underrepresented on the X (Reinke et al., 2000, 2004), although essential ovary-expressed genes tend not to be X-linked (Maeda et al., 2001; Piano et al., 2000, 2002). A trend away from germ line-expressed genes on the X was borne out by subsequent genetic studies showing that, among sets of duplicated genes, those that are expressed in the germ line tend to be located on autosomes while those expressed in the soma may be located on the X chromosome (Maciejowski et al., 2005; Ohmachi et al., 2002). Taken together, these data are consistent with the pattern of H3K4me2 and other activation marks observed by Kelly et al. (2002) in the germ line: there is little X chromosome expression during mitosis, male meiosis, and spermatogenesis, but there is a burst of X-linked expression in oogenesis.

In an initial attempt to determine the functional significance of the observed patterns of dynamic chromatin modifications, Kelly et al. (2002) compared the average transcript level for all genes versus oogenesis-expressed genes on each chromosome. They found that genes whose expression remains high during meiosis tend to be located on autosomes. In contrast, the average X chromosome transcript levels were two- to threefold lower than autosomal transcript levels in the germ line. In the soma, no significant difference in autosomal versus X chromosomal transcript level was observed. These data were consistent with X-linked transcription occurring in only a small subset of germ cells. Consistent with this hypothesis, when in situ hybridization analysis was used to visualize transcript distributions, X-linked transcripts were observed during the late-pachytene/diplotene window (Kelly et al., 2002). For each gene examined, mRNA was first visible in late pachytene nuclei, consistent with the appearance of chromatin activation marks on the X chromosomes at that stage.

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Canine Genetics

E.A. Ostrander, R.K. Wayne, in Encyclopedia of Genetics, 2001

Linkage Analyses and Genetic Maps

While chromosome gene maps are necessary for determining the evolutionary relationship between genomes, and for determining the syntenic relationships between mammals, a genetic map is needed for identifying loci which contribute to traits of interest. A genetic map is one for which the distance between markers is measured as a function of genetic recombination. A marker is a short segment of DNA that varies between homologous chromosomes in the population. Because any given individual has two copies of each chromosome, each individual must have, by definition, two alleles for every marker. If identical alleles are inherited from each parent, individuals are homozygous for that marker. Markers are considered informative if there are sufficient alleles in the population that most couplings allow the inheritance of chromosomes (or regions of chromosomes) to be tracked from grandparent to parent to offspring. If the frequency of the most common allele that appears in the population is less than 95%, then the marker is referred to as polymorphic.

If a marker and a gene are physically located close together on the same chromosome, alleles on homologous chromosomes will be coinherited in a significant number of offspring and are thus linked. If two markers are located far apart on the same chromosome or on different chromosomes their alleles will be inherited independently or randomly in offspring and are unlinked. For a given region of the genome the probability of a genetic recombination event occurring between a pair of markers or a marker and a disease gene is proportional to the distance between them. This probability is expressed as a recombination fraction or, in units called centiMorgans (cM). One percent recombination is equal to 1 cM, which roughly corresponds to a million base pairs in the human genome.

To map the gene for a trait of interest, a genomic screen of DNA from families with the trait of interest is undertaken, using markers spaced about every 5–10 cM. Figure 2 shows a schematic of a two-generation pedigree and a denaturing sequencing gel resulting from analysis of a single marker. The black bars represent alleles separated on a gel, and demonstrate Mendelian inheritance of the alleles. One allele from the father, which is circled, appears in all affected individuals. In addition, no unaffected individuals inherit this allele. Thus, it can be hypothesized that the marker indicated is close to the disease gene. Additional markers and many more families would need to be analyzed to determine if the proposed linkage is true and to determine the distance between the marker and disease gene. Odds of 1000:1 that a given marker is linked to a trait of interest are indicated by a Lod score of ≥3.0 and is generally accepted as evidence of linkage. A Lod score of less than −2.0 indicates that a given marker and trait of interest are unlinked

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Figure 2. (A) Segregation of alleles in a single pedigree. Females are represented as circles, males are squares. Affected individuals are colored in black, unaffected in white. (B) The marker analyzed here is hypothesized to be linked to the disease gene in question, since all affected individuals have inherited this allele from their affected parent or grandparent.

Currently most screens utilize genetic maps composed of microsatellite markers. Microsatellites are small repetitive stretches of polymorphic DNA that can be tracked using the polymerase chain reactions (PCR). They are optimal for the construction of genetic maps for several reasons. First, they are frequent and distributed randomly; there are several thousand of the common repeat arrays (e.g., (CA)n, (GATA)n, or (CAG)n) scattered throughout the canine genome. Hence collection of large numbers of markers for map building is a relatively straightforward exercise. Second, the rate at which mutations generate new variation/length alleles is nontrivial – about 10−5 for (CA) repeats and about 10−2 for microsatellites based upon tetranucleotide repeats. This means that they are highly informative in mapping studies in relatively inbred families. Nevertheless, they are sufficiently stable that the inheritance of adjacent sections of chromosomes can be tracked through several generations of a family with reliability.

Linkage analyses of large numbers of microsatellite markers on outbred reference families, comprised of many distinct dog breeds, have led to the production of a preliminary canine genetic map. A high-density map appears well on its way to completion, with well spaced, highly informative markers spanning several chromosomes. The map likely covers greater than 85% of the canine genome, although exact estimates are difficult to determine since the precise size of the canine genome is not known. The best estimates suggest that it is about 26.5±1.1 Morgans (95% confidence interval=24.3 M to 28.7 M). As the density and coverage of the map increases, the ability to identify loci through linkage analyses of families with traits of interest will increase proportionately. Thus far, several hundred canine microsatellites have been described and placed on the canine map (Mellersh et al., 1997), with several hundred more currently in progress. While there often appears to be a unique distribution of alleles within particular breeds, it has not yet been possible to define markers which are breed specific. This is not surprising given the discussion above about the significant genetic variation that contributed to the canine gene pool.

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Toll-Like Receptors in SLE

Terry K. Means, in Systemic Lupus Erythematosus (Fifth Edition), 2011

TLR9 Polymorphisms and Copy Number in SLE

The TLR9 gene (chromosome 3p21.3) is located in one of the defined susceptibility regions for SLE. Several groups have investigated whether genetic variations of TLR9, which include (–1486 T→C, –1237 C→T, +1174 A→G, and +2848 G→A), are involved in susceptibility to SLE in different populations around the world. The presence of the nucleotide G at position +1174 in intron 1 of TLR9 was demonstrated to be significantly associated with an increased risk of SLE in a Japanese cohort. Interestingly, the A→G +1174 SNP down-regulates TLR9 expression in reporter assays. These data would fit with the current model (discussed above) that TLR9 expression is protective against SLE.

In contrast, other studies performed on American, UK, Korean, and Chinese SLE cohorts did not detect a statistically significant association of any TLR9 gene variations with SLE susceptibility.

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The ATP7B Gene

Richard Kirk, in Clinical and Translational Perspectives on WILSON DISEASE, 2019


The ATP7B gene (chromosome 13q14.13) spans approximately 80 kb of genomic DNA, comprises 21 protein-coding exons, encodes a messenger ribonucleic acid of approximately 7.5–8.5 kb, and produces a protein containing 1465 amino acid residues. Metal-binding transcription factors may play an important role in regulation. Genetic analysis must suit the spectrum of pathogenic variants present in the population: genotyping for common mutations and/or sequencing to identify rare or novel pathogenic variants. Other rare mutational mechanisms also occur: large-scale deletions, promoter mutations, uniparental disomy, and pseudo-dominant inheritance. Diagnostic laboratories use additional quality control systems to ensure high quality of service. Approximately 800 pathogenic variants have been published. Distinguishing between pathogenic and benign variants requires the use of multiple lines of evidence. In the United Kingdom, sequencing of the protein-coding region of the ATP7B gene, with promoter sequencing and exon deletion/duplication analysis, has a 98% pickup rate for identifying significant gene alterations. The disease prevalence may be significantly higher than 1:30,000.

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Toll-Like Receptors, Systemic Lupus Erythematosus

William F. PendergraftIII, Terry K. Means, in Systemic Lupus Erythematosus, 2016

TLR9 Polymorphisms and Copy Number in SLE

The TLR9 gene (chromosome 3p21.3) is located in one of the defined susceptibility regions for SLE. Several groups have investigated whether genetic variations of TLR9, which include (−1486 T → C, –1237 C → T, +1174 A → G, and +2848 G → A), are involved in susceptibility to SLE in different populations around the world. The presence of the nucleotide G at position +1174 in intron 1 of TLR9 was demonstrated to be significantly associated with an increased risk of SLE in a Japanese cohort.50 Interestingly, the A → G +1174 SNP downregulates TLR9 expression in reporter assays. These data would fit with the current model (discussed above) that TLR9 expression is protective against SLE. A significant association was also made with −1486 T → C in an SLE cohort in China and +1174 A → G in Brazil.51,52 In contrast, other studies performed on American, British, Danish, Korean, Chinese, and Indian SLE cohorts did not detect a statistically significant association of any TLR9 gene variations with SLE susceptibility.53 In 2012, two separate meta-analyses were performed to determine the risk of SLE with three TLR9 polymorphisms (−1486 C → T, +1174 A → G, and +1635 C → T) in Asians, and no association was identified in either report.54 To date, there has yet to be a proven and replicated association of TLR9 polymorphisms with SLE risk.

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Genes, Genomes, and Genomics

Padma Nambisan, in An Introduction to Ethical, Safety and Intellectual Property Rights Issues in Biotechnology, 2017

1.7.2 Genetic Testing and Diagnostics

Genetic analysis in humans has over time revealed a number of disorders that are inherited. Documentation of this information was published between 1966 and 1998 in 12 editions of Mendelian Inheritance in Man by Victor McKusick of Johns Hopkins University School of Medicine (McKusick, 1966, 1994). Since 1987, the information became available online under the direction of the Welch Medical Library with financial support from the NCBI (McKusick, 2007). Since 2010, the website of Online Mendelian Inheritance in Man (http://www.omim.org/) is authored and edited at the McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, with financial support from the National Human Genome Research Institute (NHGRI).

Changes in chromosomes, genes, or proteins correlated to disease conditions have been used to confirm or rule out a genetic condition. Since the 1950s, amniocentesis (studying free floating cells isolated from the amniotic fluid that surrounds the fetus in the womb) has been used by physicians to diagnose chromosomal aberrations, such as Down's syndrome, in the unborn fetus. More than thousand genetic tests have been developed and can be broadly classified into three categories:

Molecular genetic tests (or gene tests),

Chromosomal genetic tests, and

Biochemical genetic tests that study the amount or activity level of proteins.

In addition to diagnosis, genetic testing can be used to identify carriers [individuals who have only one copy of the mutated gene hence do not show symptoms (phenotype) of the genetic disorder], and to determine if an individual has an inherited predisposition to a disease. The advantages of genetic testing are:

To provide an accurate diagnosis of a disease condition and to make informed decisions regarding the treatment: Genetic testing can help to identify the cause of the diseased condition and to select the appropriate treatment regimen (including whether a patient would respond to a particular treatment option). This has been found to be especially useful in the treatment of certain cancers as the therapy is expensive (thereby posing a financial burden on the patient), and could cause adverse effects (thereby further reducing the quality of life of the patient).

To make life choices: Amniocentesis has helped to make decisions regarding the medical termination of pregnancy in cases of numerical/structural abnormalities in chromosomes known to cause disease syndromes in humans. With the development of In Vitro Fertilization techniques, in which the embryo is created in a petri dish and later implanted into the womb, genetic testing is being increasingly used to select "healthy" embryos. Preimplantation Genetic Testing is especially advocated in instances when one of the biological parents is known to be affected by (or is a carrier of) a genetic disorder.

To make life-style choices: Several genetic disorders are known to be age-dependent and the disease is often manifested only after the reproductive age of the patient, hence is transmitted to the progeny (example, Huntington's disease). Genetic testing could help make informed decisions regarding reproductive choices. It could also help to plan for future healthcare and end-of-life decisions for debilitating diseases such as Alzheimer's, Parkinson's, and some forms of dementia.

Due to the rather sensitive nature of the information revealed by the tests, genetic testing is invariably coupled with genetic counseling to help patients understand how the test results would affect them and to guide them to make informed choices. The field however is mired in ethical conundrums. The ELSI initiative of the HGP is an attempt to address issues of privacy and psychological impacts of genome information revealed by genetic testing. Some of the ethical aspects associated with genetic testing are discussed in Chapter 7, Genetic Testing, Genetic Discrimination, and Human Rights of this book.

Several privately held personal genomics companies have been established, for example, the California based company 23andMe (https://www.23andme.com/en-int/) founded by Linda Avey, Paul Cusenza, and Anne Wojcicki in 2006 that has been offering direct to customer genetic testing since November 2007. (See International Society of Genetic Genealogy Wiki (2016) for a country-wise listing of personal genomics companies.)

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Chromatin Remodelling and Immunity

J.S. Rawlings, in Advances in Protein Chemistry and Structural Biology, 2017

4 Classical Functions of SMC Complexes

The first SMC gene, SMC1, was identified in Saccharomyces cerevisiae and was shown to be required for proper chromosome segregation (Strunnikov, Larionov, & Koshland, 1993). Later, it was discovered that this protein was part of the Cohesin complex (Losada, Hirano, & Hirano, 1998; Sumara, Vorlaufer, Gieffers, Peters, & Peters, 2000; Toth et al., 1999). This complex consists of a Smc1a/Smc3 heterodimer and two non-SMC components: a Kleisin family protein known as Scc1 (Rad21 in humans) and Scc3 (either STAG1 or STAG2 in humans) (Table 2). Cohesin acts as a molecular glue that holds sister chromatids together during the cell cycle beginning with DNA synthesis until anaphase of mitosis when one of the cohesion subunits, Scc1, is cleaved by Separase, a cysteine protease. This cleavage results in the release of Cohesin from sister chromatids, permitting chromosome segregation to occur (for review, see Peters, Tedeschi, & Schmitz, 2008). Because of its role in sister chromatid cohesion, it is not surprising that the Cohesin complex also has roles in DNA damage repair, specifically postreplicative double-strand break repair (Sjogren & Nasmyth, 2001).

In addition to a Smc2/Smc4 heterodimer, the Condensin I and Condensin II complexes contain two HEAT repeat containing proteins and one Kleisin subunit (Table 2). Although first discovered in 1982 as a chromosome scaffolding protein (then termed ScII), Smc2 was not characterized as a Smc protein until 1994 (Lewis & Laemmli, 1982; Saitoh, Goldberg, Wood, & Earnshaw, 1994). By 1997, the other subunits of the Condensin I complex were discovered and the complex itself was described in Xenopus egg extracts (Hirano, Kobayashi, & Hirano, 1997). The Condensin I complex functions to condense chromosomes during mitosis and is also required for proper chromosome segregation. The function of the Condensin I complex is regulated, in part, by the fact that the complex is sequestered in the cytosol during interphase. Once the nuclear envelope breaks down during prophase, the Condensin I complex can access DNA and condense it in preparation for completion of mitosis (Hirota, Gerlich, Koch, Ellenberg, & Peters, 2004; Ono, Fang, Spector, & Hirano, 2004). A second Condensin complex, found only in higher eukaryotes, was discovered more recently (Ono et al., 2003; Yeong et al., 2003). This complex, termed Condensin II, possesses the same Smc2/Smc4 heterodimer as the Condensin I complex; however, utilizes different non-SMC components (Table 2). It was immediately discovered that Condensin I and Condensin II bind different regions of chromosomes, suggesting that they contribute to chromatin architecture in distinct ways (Ono et al., 2003). Like Condensin I, Condensin II also functions in chromatin compaction and in segregation; however, unlike Condensin I, Condensin II can be found in the nucleus during interphase (Hirota et al., 2004; Ono et al., 2004). This observation signaled the possibility that higher-order chromosome condensation mediated by Condensin II is not limited to cell division and that Condensin II could play roles in interphase biology in higher eukaryotes.

The third Condensin complex, termed Condensin IDC, is a part of the dosage compensation complex (DCC) found only in Caenorhabditits elegans and unlike the other Condensins, it contains a heterodimer of DPY-27 and Smc2. As its name implies, the primary role of the DCC is to mediate dosage compensation of X chromosome genes in hermaphrodites (Chuang, Albertson, & Meyer, 1994; Csankovszki et al., 2009; Lieb, Albrecht, Chuang, & Meyer, 1998; Lieb, Capowski, Meneely, & Meyer, 1996; Tsai et al., 2008). Unlike dosage compensation in mammals which is achieved by the random silencing of a single X chromosome, the DCC interacts with both X chromosomes to downregulate gene expression by half (Ercan & Lieb, 2009; Heard & Disteche, 2006).

The Smc5/6 complex is perhaps the least understood of the SMC complexes. The first component of this complex (Smc6) was originally identified because it was able to rescue a radiation-sensitive mutant strain of Schizosaccharomyces pombe (Nasim & Smith, 1975; Phipps, Nasim, & Miller, 1985). In addition to the Smc5/6 heterodimer, this complex contains four non-SMC components. Sequence analysis of these subunits reveals that the complex may have E3 ubiquitin ligase activity as well as SUMO ligase activity (Lehmann, 2005). Other than its known roles in DNA repair, the functions of the Smc5/6 complex remain elusive (Lehmann et al., 1995; Verkade, Bugg, Lindsay, Carr, & O'Connell, 1999). However, recent work suggests that there is at least crosstalk between the Smc5/6 complex and the Cohesin complex during chromosome segregation (for review, see Tapia-Alveal, Lin, & O'Connell, 2014).

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Normal and Aberrant Growth in Children

David W. Cooke, ... Sally Radovick, in Williams Textbook of Endocrinology (Thirteenth Edition), 2016


The POU1F1 gene (chromosome 3p11, OMIM 173110) encodes PIT1, a member of a large family of transcription factors referred to as POU-domain proteins that is responsible for pituitary-specific transcription of genes for GH, PRL, TSH, and the GHRH receptor.58,454-456 PIT1, a 290–amino acid protein, contains two domains, the POU-specific and the POU-homeo domains; both are necessary for DNA binding and activation of GH and PRL genes and for regulation of the PRL, TSH-β, and POU1F1 genes.457 Its expression is restricted to the anterior pituitary to control differentiation, proliferation, and survival of somatotrophs, lactotrophs, and thyrotrophs.371,456-458 PIT1 regulates target genes by binding to response elements and recruiting coactivator proteins, such as cAMP response element–binding protein (CREB)-binding protein (CBP).459 Gene expression microarray assays combined with chromatin immunoprecipitation (CHIP) were used to detect targets of POUIFI.273

Two mouse models were first reported to have GH, PRL, and TSH deficiencies associated with mutations or rearrangements of the Pit1 gene; these were the Snell (dw/dwS) and the Jackson (dw/dwJ) dwarf mice.460,461 Many different mutations of the POU1F1 gene have been found internationally in families with GHD and PRL deficiency and variable defects in TSH expression.462-465 These mutations are transmitted as autosomal recessive or dominant traits and cause variable peptide hormone deficiencies with or without anterior pituitary hypoplasia.462-468

The most common mutation is an R271W substitution that affects the POU homeodomain, encoding a mutant protein that binds normally to DNA and acts as a dominant inhibitor of transcription.468-471 Vertical transmission of the R271W mutation was shown, emphasizing the importance of diagnostic and theraputic management during pregnancy.472 Evidence from a patient with the R271W mutation suggests that PIT1 may have a role in cell survival.471 Indeed, the mutation was used to target cell proliferation tumoral model systems. A patient diagnosed with GHD, along with dysregulation of PRL and TSH, was reported to have a lysine-to–glutamic acid mutation at codon 216 (K216E).457 This mutant PIT1 binds to DNA and appears not to inhibit basal activation of GH and PRL genes; however, the mutant is unable to support retinoic acid induction of POU1F1 gene expression. Another report suggested that CBP (p300) recruitment and PIT1 dimerization are necessary for target gene activation and that disruption of this process may account for the pathogenesis of CPHD.473 All of the reported mutations involve sites affecting POU1F1 DNA-binding, dimerization, or target gene transactivation.

Phenotypic variability occurs among patients with apparently similar genotypes. It does not appear that ACTH or gonadotropin deficiencies occur, as is frequently the case with PROP1 defects,452 but adrenarche has been reported to be absent or delayed in patients with a POU1F1 mutation.474 Circulating antibodies against Pit1 have been identified to be responsible for hypopituitarism similar to that caused by mutations.475

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Biochemistry and Molecular Biology

K.J. Kramer, S. Muthukrishnan, in Comprehensive Molecular Insect Science, 2005 Regulation of chitin synthase gene expression

The two insect genes encoding CHSs appear to have different patterns of expression during development. The high degree of sequence identity of the catalytic domains and the absence of antibodies capable of discriminating between the two isoforms have complicated the interpretation of experimental data to some extent. In some cases, the technical difficulties associated with isolation of specific tissues free of other contaminating tissues have precluded unambiguous assignment of tissue specificity of expression. Nonetheless, the following conclusions can be reached from the analyses of expression of CHS genes in several insect species. CHS genes are expressed at all stages of insect growth including embryonic, larval, pupal, and adult stages. CHS1 genes (coding for class A CHS proteins) are expressed over a wider range of developmental stages (Tellam et al., 2000; Zhu et al., 2002). CHS2 genes (coding for class B CHSs) are not expressed in the embryonic or pupal stages but are expressed in the larval stages, especially during feeding in the last instar and in the adults including blood-fed mosquitoes (Ibrahim et al., 2000; Zimoch and Merzendorfer, 2002; Arakane et al., 2004). The finding that both classes of CHS genes are expressed at high levels 3 h after pupariation in Drosophila suggests that both enzymes are required for postpuparial development (Gagou et al., 2002).

CHS genes also show tissue-specific expression patterns. In L. cuprina, CHS1 (coding for a class A CHS) is expressed only in the carcass (larva minus internal tissues) and trachea but not in salivary gland, crop, cardia, midgut or hindgut (Tellam et al., 2000). In blood-fed female mosquitoes, a CHS gene encoding a class B CHS is expressed in the epithelial cells of the midgut (Ibrahim et al., 2000). In M. sexta, CHS1 (coding for a class A CHS) is expressed in the epidermal cells of larvae and pupae (Zhu et al., 2002). Transcripts specific for class B CHS were detected only in the gut tissue (D. Hogenkamp et al., unpublished data). As discussed above, in Drosophila, both classes of CHS genes were shown to be upregulated after the ecdysone pulse had ceased in the last larval instar, but the tissue specificity of expression of each gene was not determined. In T. castaneum, the CHS1 gene (coding for a class A CHS) was expressed in embryos, larvae and pupae, and in young adults, but not in mature adults (Arakane et al., 2004). Even though unequivocal data are not available for each of these insect species, the following generalizations may be made. Class A CHS proteins are synthesized by epidermal cells when cuticle deposition occurs in embryos, larvae, pupae, and young adults, whereas the class B CHS proteins are expressed by the midgut columnar epithelial cells facing the gut lumen in the larval and adult stages and is probably limited to feeding stages.

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SDIS enables exchange and comparison of DNA profiles within a state and is usually operated by the agency responsible for maintaining a state's convicted offender DNA database program.

From: Fundamentals of Forensic DNA Typing, 2010

Related terms:


Law Enforcement





Research Workers

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Alan Sandomir, John Butler, in **** Investigation Handbook (Second Edition), 2011

Access to CODIS

The CODIS program is usually maintained at the Local DNA Index System (LDIS), State DNA Index System (SDIS), and National DNA Index System (NDIS) levels. By 1998, all 50 states, Puerto Rico, and the District of Columbia had passed legislation allowing for the collection of DNA samples from specified convicted offenders. However, this does not preclude local police labs or coroner/medical examiner offices from maintaining their own DNA data bank of nonconvicted offenders or suspects (through the LDIS; although this DNA group may not be necessarily compared at the state or national CODIS levels, as some of the subjects are not convicted).

Police labs, medical examiner/coroner labs, and private labs utilized by law enforcement began to develop and compare these DNA fingerprints or "profiles" within their jurisdictions. After being compared at the local and state levels, these profiles are uploaded into the FBI's CODIS (NDIS) for a nationwide search and comparison. These profiles, at the LDIS, SDIS, and NDIS levels, have churned out a wealth of information linking crimes and suspects by DNA. Not only have local and state law enforcement agencies been receiving DNA case links and matches from within their own states, but the national CODIS has linked and matched cases across state lines and across the country.

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DNA Amplification (The Polymerase Chain Reaction)

John M. Butler, in Fundamentals of Forensic DNA Typing, 2010

Stochastic effects from low levels of DNA template

Forensic DNA specimens often possess low levels of DNA. When amplifying very low levels of DNA template, a phenomenon known as stochastic fluctuation can occur. Stochastic effects, which are an unequal sampling of the two alleles present from a heterozygous individual, result when only a few DNA molecules are used to initiate PCR. PCR reactions involving DNA template levels below approximately 100 pg of DNA, or about 15 diploid copies of genomic DNA, have been shown to exhibit allele dropout. False homozygosity results if one of the alleles fails to be detected.

Stochastic artifacts can be avoided by adjusting the cycle number of the PCR reaction such that approximately 20 or more copies of target DNA are required to yield a successful typing result. However, efforts have been made to obtain results with low copy number (LCN) DNA testing. The challenges of LCN work and trying to interpret data obtained with less than 100 pg of DNA template will be addressed in Chapter 14 and the Advanced Topics volume. Whole genome amplification prior to PCR and locus-specific amplification also have the same issues in terms of stochastic fluctuations with low levels of DNA (D.N.A. Box 7.1).

D.N.A. Box 7.1

Whole Genome Amplification


Ballantyne, K. N., et al. (2006) Molecular crowding increases the amplification success of multiple displacement amplification and short tandem repeat genotyping. Analytical Biochemistry, 355, 298-303.



Schneider, P. M., et al. (2004). Whole genome amplification—The solution for a common problem in forensic casework?.Progress in Forensic Genetics 10 ICS 1261, 24-26.

A common challenge with forensic casework is the recovery of limited quantities of DNA from evidentiary samples. Within the past few years a new DNA enrichment technology has been developed known as whole genome amplification (WGA). WGA involves a different DNA polymerase than the TaqGold enzyme commonly used in forensic DNA analysis. WGA amplifies the entire genome using random hexamers as priming points. The WGA enzymes work by multiple displacement amplification (MDA), which is sometimes referred to as rolling circle amplification. MDA is isothermal with an incubation temperature of 30°C and requires no heating and cooling like PCR.

Qiagen (Valencia, CA) and Sigma-Aldrich (St. Louis, MO) both offer phi29 DNA polymerase cocktails for performing WGA. The kit sold by Qiagen is called REPLI-g while Sigma-Aldrich's kit is GenomePlex. Yields of 4–7 μg of amplified genomic DNA are possible from as little as 1 ng of starting material. The phi29 enzyme has a high processivity and can amplify fragments of up to 100 kb because it displaces downstream product strands, enabling multiple concurrent and overlapping rounds of amplification. In addition, phi29 has a higher replication fidelity compared to Taq polymerase due to 3′–5′ proofreading activity.

While all of these characteristics make WGA seem like a possible solution to the forensic problem of limited DNA starting material, studies have found that stochastic effects at low levels of DNA template prevent WGA from working reliably (Schneider et al., 2004). Allele dropouts from STR loci were observed at 50- and 5-pg levels of starting material (Schneider et al., 2004) just as are seen with current low copy number DNA testing (see Chapter 14). Work with 'molecular crowding' materials such as polyethylene glycol, where the amount of DNA is enriched in localized areas of a sample, has shown improved success with STR typing from low amounts of DNA (Ballantyne et al., 2006). While it is possible that WGA may play a limited role in enriching samples for archiving purposes that are in the low nanogram range, it will probably not be the end-all solution to low copy number DNA samples.

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DNA Databases

John M. Butler, in Fundamentals of Forensic DNA Typing, 2010

Missing persons

DNA databases can also play an important role in helping to identify missing individuals and aiding mass disaster reconstruction following a plane crash or terrorist activity (see Chapter 17). In these cases, DNA samples are often obtained from biological relatives that can be searched against DNA of remains recovered from a missing individual or a disaster site. Many states within the United States and nations around the world are beginning to establish missing persons databases to enable matching of recovered remains to their family members.

CODIS also has several indices to aid missing-person investigations that can store DNA profiles from both recovered remains and family samples that serve as references. Much of the data from missing-person investigations is in the form of mitochondrial DNA sequences since this information can be successfully recovered from highly degraded samples. As noted in Chapter 16, use of mitochondrial DNA also enables access to a larger number of reference samples from maternal relatives of a victim.

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Biometrics in the Criminal Justice System and Society Today

Dr.Thomas J. Rzemyk Ed.D., CHPP, CAS, in Effective Physical Security (Fifth Edition), 2017

Deoxyribonucleic Acid

DNA is perhaps one of the most important biometrics today as it has been used to solve thousands of crimes around the globe. The technology surrounding DNA is always evolving and new enhancements are being applied to the law enforcement community. One of the most recent improvements has been the development of the Rapid DNA Program Office established in 2010 by the FBI. Rapid DNA, or Rapid DNA Analysis, describes the fully automated (hands-free) process of developing a CODIS Core STR profile from a reference sample buccal swab. The "swab in—profile out" process consists of automated extraction, amplification, separation, detection, and allele calling without human intervention.8 The FBI's Imitative is to improve the process and the time that it takes to complete DNA testing by integrating technologies into CODIS and other DNA-related systems. In sum, the benefit of using DNA as a biometric identifier is the level of accuracy offered. Similar to fingerprint data, it is nearly impossible for two human subjects to share the same DNA structure.

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Nutrients and Aging

Lawrence J. Whalley, in Handbook of Models for Human Aging, 2006

DNA-protective nutrients

DNA-protective nutrients are present in diet. This is best regarded as a mixture of compounds, some of which are potentially harmful to DNA, whereas others are protective. The overall effect of diet on the accumulation of damaged DNA reflects the sum of their opposing actions. Nutrients that protect DNA include the antioxidants vitamin C and vitamin E, and the carotenoids, probably by stabilizing free radicals. All plant leaves contain chlorophyll, which forms a water-soluble salt, chlorophyllin, that can protect DNA from damage. This last mechanism is an example of those many biological processes (open to genetic influence) that may activate or inhibit conversion of dietary compounds to potent DNA-damaging agents. So far, none of these processes are studied extensively in aging populations, so their general importance, though recognized, remains unknown. The underlying biology, though complex, suggests pathways that may modify the rate of progression in aging and on to age-related diseases.

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Choline and Brain Development

Mihai D. Niculescu, in Nutrition in the Prevention and Treatment of Disease (Fourth Edition), 2017

a Epigenetic Mechanisms Regulate Gene Expression

DNA methylation is represented by the substitution of hydrogen with methyl groups to DNA (reviewed in [117]). In mammalians, the majority of DNA methylation occurs at carbon 5 of the cytosine ring (5-methylcytosine; 5mC) only when the cytosine is followed by a guanine nucleotide (CpG site), but methyl groups can be also added to other nucleotides [117]. This process is catalyzed by DNA methyltransferases (DNMTs). During the S-phase of cell-cycle progression, the DNA methylation status of the parental DNA strand is duplicated on the newly synthesized DNA by maintenance DNA methyltransferase, DNMT1 [118]. However, DNA methylation can also occur at previously unmethylated CpG sites (de novo DNA methylation), catalyzed by de novo DNA methyltransferases, DNMT3a and 3b, and with participation of DNMT2 and 3L [118].

When DNA methylation occurs within promoter regions, it usually associates with gene underexpression and chromatin compaction [51], but instances have been described in which promoter hypermethylation prevented the binding of inhibitory factors, thus allowing for promoter activation and gene overexpression [119]. The establishment of cell type-specific DNA methylation patterns contributes decisively to shaping the cellular phenotypes of differentiated cells [118].

Within the concept of epigenetic regulation of gene expression, an important feature is genomic imprinting, allowing genes to be expressed in a parent-of-origin manner (imprinted genes), and this process being the molecular basis for monoallelic expression [120]. During early embryogenesis, most of the parental DNA methylation patterns are erased by active and passive demethylation mechanisms (with the exception of some imprinted regions), whereas new DNA methylation patterns are established by de novo methylation. The establishment of new epigenetic patterns continues during fetal morphogenesis and in the early postnatal period [121].

The epigenetics of DNA also includes the hydroxylation of methyl groups attached to cytosine, as an intermediary step in active DNA demethylation, with important functional consequences on gene activation [122]. This groundbreaking discovery provided the first plausible mechanism for the previously observed active DNA demethylation [116].

Chromatin modifications occur at the flexible tail regions of histones. These modifications include, but are not limited to, methylation, acetylation, phosphorylation, ubiquitination, and ADP ribosylation [123]. In concert with DNA methylation, histone modifications allow for the reversible switch between chromatin relaxation and compaction and also the establishment of the degree of access that transcription factors have to promoter regions [118,123]. Examples are methylation of histone H3 at its lysine 9 and 27 residues (K9 and K27), allowing for chromatin compaction and inhibition of gene expression, and trimethylation of H3K4 that induces transcriptional activation and promoter activation [123].

MicroRNAs (miRNAs) are noncoding RNA species, up to 25 nucleotides in length, that contribute to gene expression regulation through RNA interference [124]. Their epigenetic role consists of the modulation of expression for several genes involved in the epigenetic machinery, which are responsible for DNA and histone modifications (e.g., DNMT3a/b, HDAC1/4, and MeCP2) [124]. Some genes encoding miRNA species can also be epigenetically regulated because their gene expression is highly dependent on their promoter methylation (reviewed in [124]).

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Gene Editing in Regenerative Medicine

Yunlan Fang, ... W.T. Godbey, in Principles of Regenerative Medicine (Third Edition), 2019


DNA is often delivered as a plasmid, which is typically constructed via standard molecular cloning techniques. When properly preserved, DNA has a long shelf life. The efficiency of DNA delivery into cells is the major limiting factor for the associated methods. DNA for genome editing can also be engineered into a viral genome, and plasmids can be adapted for use with viruses for delivery into cells [33]. Although viruses typically generate higher gene delivery efficiencies than do nonviral gene delivery agents, immunogenicity is a concern for in vivo applications, especially if repeated delivery events must be performed.

Another concern regarding the delivery of DNA into cells is that the foreign DNA could integrate into the host genome through homologous recombination. If this occurs in an untargeted fashion, vital host genes may be disrupted or inactivated, tumor suppressor genes may be knocked out, or oncogenes might be activated. Whether targeted or untargeted, genomic integration could cause the encoded nuclease to be expressed in a sustained manner, potentially causing continuous formation of double-strand breaks and serious off-target effects.

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Liquid crystalline DNA

Luciano De Sio, ... Roberto Bartolino, in Hybrid Polymer Composite Materials, Volume 4, 2017

13.1 Deoxyribonucleic acid

13.1.1 DNA and its liquid crystalline phases

DNA is a semiflexible polymer made of two polynucleotide chains, held together by weak thermodynamic forces (Lander and Weinberg, 2000). The monomer units of DNA are the nucleotides. Each nucleotide consists of a 5-carbon sugar (deoxyribose), a nitrogen containing base attached to the sugar, and a phosphate group (Travers and Muskhelishvili, 2015). There are four different types of nucleotides found in DNA: adenine (A), thymine (T), cytosine (C), and guanine (G). A and T are connected by two hydrogen bonds while G and C are connected by three hydrogen bonds (Fig. 13.1).

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Figure 13.1. DNA chemical structure.

An interesting and comprehensively studied phenomenon is represented by the tendency of flexible polymers in concentrated aqueous solutions to form liquid crystalline phases (Onsager, 1949; Nakata et al., 2007). Indeed, the ability of both long and ultrashort, hydrated, double-stranded DNA molecules to form liquid crystal (LC) phases has been known for more than 50 years and played a key role in the initial deciphering of the molecular DNA. Linear DNA fragments in aqueous solution form multiple LC phases whose nature depends on the polymer concentration: when increasing concentration, the isotropic solution transforms into either blue phase or precholesteric stage and then into a cholesteric phase which turns itself into columnar hexagonal (Strzelecka et al., 1988).

13.1.2 DNA extraction

The isolated DNA can be obtained by using the Wizard Genomic DNA Purification Kit, designed to extract DNA from different sources, such as white blood cells, tissue culture cells, animal tissue, and others. In our specific case, we have chosen to follow the DNA extraction method starting from fresh whole human blood. The sample of whole blood was collected in an EDTA anticoagulant tube to prevent the clotting and kept at −20°C. For the utilization, the frozen blood sample was thawed out and left at room temperature (3–4 h) before starting the DNA isolation method. The DNA extraction method is illustrated in Fig. 13.2:

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Figure 13.2. Sketch of the DNA extraction method.


A volume of 300 μL of blood was put in a sterile 1.5 mL microcentrifuge tube which contained 900 μL of cell lysis solution. The mixture was mixed by inverting the tube 5–6 times and was incubated for 10 min at room temperature to lyse the red blood cells. The mixture was then centrifuged for 50 s (14,000×g) at room temperature. Supernatant was removed and discarded as much as possible without disturbing the visible white pellet. To obtain it, an additional aliquot of Cell Lysis Solution was added and the incubation and centrifugation steps were repeated. The pellet was resuspended by a brief vortexing.


The white blood cells and their nuclei (where the DNA is accumulated) underwent lysing. Nuclei lysis solution (300 μL for 300 μL sample volume) was added to the tube containing the resuspended cells and the mixture was again incubated for 1 h at 37°C. At this point, a RNase solution was added (1.5 μL for 300 μL sample volume) to the nuclear lysate; the sample was mixed by inverting the tube 2–5 times. The mixture was incubated at 37°C for 15 min and then cooled down to room temperature.


The cellular proteins were removed by means of a salt precipitation. Protein precipitation solution was added (100 μL for 300 μL sample volume) to the nuclear lysate, after the mixture was centrifugated for 3 min (14,000×g) at room temperature. In this way, the cellular proteins were removed while the high molecular weight genomic DNA was left in the solution.


The supernatant was transferred to a clean tube containing 300 μL of isopropanol. Therefore, the solution was gently mixed by inversion until the macromolecules of DNA formed a visible mass. After centrifugation for 1 min (14,000×g) at room temperature, the DNA was visible as a small white pellet. The pellet was washed with 70% ethanol. After centrifugation for 1 min (14,000×g) at room temperature, the DNA pellet and the sides of the tube were gently washed. By using a drawn Pasteur pipette, the ethanol was carefully aspired. The tube was inverted on a clean absorbent paper and the pellet was air-dried for 1–2 h. Finally, the DNA was resuspended in 100 μL of rehydration solution and incubated at 65°C for 1 h.

13.1.3 Agarose gel electrophoresis analysis

It is a technique particularly suited for accomplishing both separation and analysis of macromolecules (e.g., DNA, RNA, proteins) and their fragments, based on their length and charge (Lee et al., 2012). Charged molecules of the solution to be tested are forced to move through an agarose matrix under the influence of an electric field that is created by applying an external DC voltage. The DNA molecules, being negatively charged along their length, migrate toward the positive electrode. In this way, during their electrophoretic run, they are separated according to their length, since pores in the agarose matrix allow short DNA molecules to snake through more easily than long ones.

13.1.4 DNA labeling

Labeling is a basic technique of molecular biology that allows to determine the position of a particular nucleic acid molecule on a membrane or gel, on a chromosome or within a tissue or a cell; the emitted signal can be detected appropriately by fluorescence microscopy (Bellizzi et al., 2007). DNA molecules can be easily and optimally labeled with radioactive phosphates, biotin, fluorophores, and enzymes during their synthesis. Nonisotopic DNA labeling procedures are essential for integration of DNA diagnostics into the clinical laboratory. Labeling DNA can be performed at either the 5′ or 3′ ends or all along the molecule, depending on the application. For applications such as in situ hybridization, northern and southern blot hybridization, it is usually advantageous to generate probes with label distributed throughout the nucleic acid; for other applications like protein interactions, end-labeled probes are preferentially generated. The main procedures for labeling a double-stranded DNA fragment are 3′ end-labeling with DNA polymerase (enzymes that create deoxyribonucleic acid polymers) or at the 5′ end using T4 polynucleotide kinase (enzymes that transfer the phosphate group of [γ32-P]ATP to the 5′ hydroxyl terminus of a DNA molecule); nick translation, using DNA polymerase in the presence of at least one radioactive precursor, to "translate" a nick (single-strand cut in the double-stranded DNA molecule) along the molecule in the 5′ to 3′ direction; random primer labeling, where the DNA molecule is used as a probe and it is denaturated by heating and mixed with random hexanucleotides which prime the polymerization reactions; PCR (polymerase chain reaction) labeling. It is recommended to purify labeled probes by many methods: spin column chromatography, membrane filtration, adsorption to silica gel membranes, ethanol precipitation.

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Fundamentals of DNA Separation and Detection

John M. Butler, in Fundamentals of Forensic DNA Typing, 2010

CE electrokinetic injection

To get DNA molecules onto the CE capillary, an electric voltage is applied while the end of the capillary is immersed into the liquid DNA sample. The flow of current generated by the voltage applied and the resistance experienced will pull the negatively charged DNA molecules onto the end of the capillary. Unfortunately, PCR products, in addition to the fluorescently labeled DNA molecules, also contain small salt ions, such as chloride, that compete with the DNA to be loaded onto the capillary. The PCR products created from amplifying a genomic DNA sample with an STR typing kit (see Chapter 8) are typically diluted to levels of approximately 1 in 10 with deionized formamide (e.g., 1 μL PCR product into 9 μL of formamide) both to help denature the double-stranded DNA molecules and to help reduce the salt levels and aid the electrokinetic injection process.

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Outline of Epigenetics

Bidisha Paul, Trygve O. Tollefsbol, in Epigenetics in Psychiatry, 2014

Chromatin remodeling and histone modifications

DNA is wrapped around spools known as histones, which are octameric proteinaceous structures. The core histones are comprised of four proteins known as H2A, H2B, H3, and H4. In histones, 147 base pairs of DNA are wrapped 1.65 times around the octamer structure and are stabilized with the assistance of linker histones known as H1. This organization is collectively referred to as chromatin. There is a very strong ionic bond between the negatively charged DNA phosphate backbone and the highly positively charged amino acids of the nucleosomes. This tight and compact structure inhibits the binding of other proteins to DNA. Not all of the DNA is bound to histones, and between every pair of nucleosomes, along the length of chromosomal DNA, are gaps referred to as linker DNA. This DNA is much more accessible to proteins and transcription factors.

There are two ways in which chromatin can be organized. Euchromatin is relatively loosely packed and hence the transcriptionally active form of organization. Heterochromatin, on the other hand, is the compact and transcriptionally inactive form of organization. The N-terminal tails of each histone protrude out and are sites for various covalent posttranslational modifications such as methylation, acetylation, phosphorylation, ubiquitination, sumoylation, and ADP ribosylation. Histone modifications can also occur in the globular domain of histones. There are two ways in which histone modifications regulate transcription. First, the modifications change the structure and orientation of the chromatin; as a result, they become more accessible to transcription factors. The other mechanism is via signals. The first modification often acts as a signal for subsequent histone modifications by recruiting specific enzymes.

Journal of Autoimmunity

Volume 38, Issues 2–3, May 2012, Pages J187-J192

The X chromosome and immune associated genes

Author links open overlay panelIlariaBianchiaPietroInvernizziac

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https://doi.org/10.1016/j.jaut.2011.11.012Get rights and content


The X chromosome is known to contain the largest number of immune-related genes of the whole human genome. For this reason, X chromosome has recently become subject of great interest and attention and numerous studies have been aimed at understanding the role of genes on the X chromosome in triggering and maintaining the autoimmune aggression. Autoimmune diseases are indeed a growing heath burden affecting cumulatively up to 10% of the general population. It is intriguing that most X-linked primary immune deficiencies carry significant autoimmune manifestations, thus illustrating the critical role played by products of single gene located on the X chromosome in the onset, function and homeostasis of the immune system. Again, the plethora of autoimmune stigmata observed in patients with Turner syndrome, a disease due to the lack of one X chromosome or the presence of major X chromosome deletions, indicate that X-linked genes play a unique and major role in autoimmunity. There have been several reports on a role of X chromosome gene dosage through inactivation or duplication in women with autoimmune diseases, for example through a higher rate of circulating cells with a single X chromosome (i.e. with X monosomy). Finally, a challenge for researchers in the coming years will be to dissect the role for the large number of X-linked microRNAs from the perspective of autoimmune disease development. Taken together, X chromosome might well constitute the common trait of the susceptibility to autoimmune diseases, other than to explain the female preponderance of these conditions. This review will focus on the available evidence on X chromosome changes and discuss their potential implications and limitations.


► The X chromosome contains the largest number of immune-related genes of human genome. ► X chromosome defects may explain the female preponderance in autoimmune diseases. ► High number of X monosomy cells was found in women with autoimmune diseases. ► X-linked genes dosage may play a role in loss of tolerance. ► X-linked microRNAs may be involved in development of autoimmune diseases.




Sex chromosomes

Genetic factors


Female preponderance




autoimmune diseases


genome-wide association study


primary biliary cirrhosis


major histocompatibility complex


primary immunodeficiency syndromes


X chromosome inactivation


systemic lupus erythematosus


systemic sclerosis


autoimmune thyroid disease


X-linked hyper-IgM syndrome


Polyendocrinopathy, and enteropathy,

Monosomy for the X chromosome in humans creates a genetic Achilles' heel for nature to deal with. We report that the human X chromosome appears to have one-third the density of the coding sequence of the autosomes and, because of partial shielding from the high mutation rate of the male sex, that it should also have a lower mutation rate than the autosomes (i.e., .73). Hence, the X chromosome should contribute one quarter (.33×.73=.24) of the deleterious mutations expected from its DNA content. In this way, selection has possibly moderated risks from mutation in X-linked genes that are thought to have been fixed in their syntenic state since the onset of the mammalian lineage. The unexpected difference in the density of coding sequences indicates that our recent, hemophilia B–based estimate of the rate of deleterious mutations per zygote should be increased from 1.3 to 4 (1.3×3).



Intuitively, the rate of mutations per genome seems likely to be determined by a trade-off between the benefits of reducing the deleterious mutation rate and the cost of increasing fidelity.

The mammalian X chromosome could make an excessive contribution to the yield of deleterious mutations because of monosomy in males. Hence, McVean and Hurst (1997) proposed that selection would favor a lower mutation rate on the X chromosome and presented supporting data from mouse- and rat-sequence divergence. In birds, however, results were obtained that were inconsistent with this hypothesis (Ellegren and Fridolfsson 1997).

The idea of chromosome-specific mutation rates is not very attractive, because DNA sequence fidelity relies on general properties of replication, damage repair, and cellular checkpoints that monitor the integrity and successful replication of DNA during mitotic or meiotic cell cycles.

Nature could moderate the X-chromosome yield of deleterious mutations in other ways—for example, by ensuring that this chromosome has a small target for deleterious mutations per megabase of DNA. Chromosomal gene content does not seem strictly proportional to size, since (1) the phenotypic effects of trisomies are not a simple function of chromosome length (Jacobs and Hassold 1995); (2) dramatic interspecies differences exist in genome size (e.g., 3×109 bp in humans and 4×108 bp in Fugu [Brenner et al. 1993]), relative to gene number; (3) chromosomes vary in the content of DNA isochores associated with gene richness (Bernardi 1989); and (4) chromosome paint for CpG islands, which mark ∼60% of genes (Antequera and Bird 1993), suggests a deficit in the human X chromosome (Craig and Bickmore 1994). These considerations prompted us to ask whether, on average, human X-linked genes show a smaller (than autosomal) ratio of coding to noncoding DNA.

We searched databases (GenBank and Integrated X Chromosome Database) for human X-linked genes with at least one autosomal homologue and known genomic and mRNA structure. Only groups of genes with identity ≥35% throughout the length of encoded proteins were considered.

Table 1 shows that a majority of X-linked genes are larger than their autosomal homologues, despite similar exon numbers. Some genes show similar base composition, and others show different levels of G+C richness. A+T-rich genes are usually larger than their G+C-rich homologues. On average, the X-linked genes available for comparison are threefold larger than their autosomal homologues. There are still few informative genes, but the X-chromosome and autosomal sequences available provide further data.

Table 1. Structure of X-Linked Genes and Their Autosomal Homologues

X-Linked GeneAutosomal HomologueNameNo. of ExonsmRNA (kb)Size (kb)Genomic G+C/mRNA G+C (%)NameNo. of ExonsmRNA (kb)Size (kb)Genomic G+C/mRNA G+C (%)AVPR31.6265/65OXYR44.118.9?/49GUCY203.710940/47GUCY2D203.612.861/65PDK3101.64040/43PDK4111.810.636/46PLS3161.7∼90?/42LCP1161.9∼90?/46F982.83439/39F1082.5∼25?/59F782.51261/59PRTC81.8958/60F8C269196?/42F5256.7∼72?/45COL4A5516.725036/50COL4A1525∼100?/60COL4A6466.4∼425?/56DMD7914∼2,400?/42UTRN?13∼900?/45SLC6133.78.665/62SLC6A11162∼25?/56SLC6A2141.945?/57TBG51.85.241/43P1441.36.148/53TIMP15.8∼3?/58TIMP251∼83?/65TIMP354.661?/49 Average4.48296.93.5898.0

The sequence of chromosome 22 was announced recently (Dunham et al. 1999), and much of the X chromosome has been sequenced. Using AceBrowser, we examined the 22.7 Mb of chromosome 22 and the 39.8 Mb of the X chromosome, sequenced and annotated using identical criteria at the Sanger Centre. This showed 752 and 1,405 exons (pseudogenes and noncoding exons were excluded) in the X-chromosome and chromosome 22 sequences (table 2). The latter showed 2.55 times more coding information per megabase than the former, in keeping with the data on the aforementioned X-linked and homologous autosomal genes. Hence, the X chromosome appears to have a lower gene density and should yield fewer deleterious mutations per megabase of DNA. This contrasts with the suggestion by Cooper and Schimtke (1984), who noted a lower level of RFLP heterozygosity on the X chromosome, relative to the autosomes, and who suggested that this might be explained by the X chromosome being particularly rich in coding and regulatory sequences.

Table 2. Coding Sequence in X-Chromosome and Chromosome-22 Regions Examined at the Sanger Centre

ChromosomeX22X:22 RatioFinished sequence (Mb)39.822.71.75No. of exons7521,405.535Sum of exon length (bp)153,603223,730.686Sequence ratio (total:exon)2591012.55

The sequence variation observed by the authors of these earlier reports is expected to be essentially neutral, and a better explanation for the low level of "neutral" sequence variations on the X chromosome arises from consideration of the joint consequences of the two following facts. There are only three X chromosomes, versus four of each type of autosome, so that a site on the X chromosome has three fourths the chance of mutating of a site on an autosome. The X chromosome spends only one-third of its time in males, versus half for the autosomes. Therefore, the overall mutation rate of the X chromosome should be lower than that of the autosomes, when the male:female ratio of mutation rates is >1. Using data from the U.K. hemophilia B population, we have directly estimated (Green et al. 1999) that this ratio is 8.6, and, consequently, the human X-chromosome mutation rate should be 27% lower than that of autosomes {[1−(2+8.6)×2/(1+8.6)×3]×100=27}. In addition, background selection against recessive deleterious mutatins will further reduce the frequency of neutral variation on the X chromosome relative to autosomes (Charlesworth 1994).

Ohno (1967) has presented compelling arguments to suggest that the development of an X-Y sex-chromosome dimorphism and the device of X inactivation to achieve dosage compensation have constrained the evolution of the X chromosome in the mammalian lineages, so that its gene composition, in contrast to that of individual autosomal pairs, has been conserved. It follows from this—now known as Ohno's law—that the X chromosome has not been able to escape the selective pressures mentioned at the outset of this report, by reducing or substantially changing its gene content during the evolution of the mammalian lineage.

The mammalian method of X-linked gene-dosage compensation results in the activity of only one X chromosome in female somatic cells from early embryonic life (Lyon 1998). It follows that, uniquely in mammals (Lucchesi 1998), even females are exposed to some risk from X-linked gene hemizygosity. This further increases the pressure of purifying selection. A small target for deleterious mutations in the X chromosome thus seems appropriate.

Lyon (1998) reviewed evidence on the spreading of X inactivation and proposed that this is favored by interspersed repetitive elements of the LINE (long interspersed repeat-sequence element) type that may have accumulated in the X chromosome under the influence of selection. Hence, a single process may have favored X inactivation and low target density for deleterious mutations.

In view of the lower density (.33) of coding sequences that we found on the X chromosome and the lower X-chromosome mutation rate resulting from the high male:female ratio of mutation rates (1−.27=.73), we propose that the rate of deleterious mutations per megabase of X chromosome should be only one quarter (.73×.33=.24) of that per autosomal megabase.

Recently, we suggested that a human zygote carries 128 new mutations, of which 1.3 are deleterious or, according to our definition, capable of causing clinically detectable effects in the hemizygous state (Giannelli et al. 1999). We based our calculations on data from X-linked hemophilia B but did not take into account the X chromosome's threefold-lower target density for deleterious mutations. Therefore, a value of four deleterious mutations (i.e., 1.3×3.3) per human zygote is more appropriate. Such a rate suggests that the continued existence of our species probably depends on quasi-truncating selection and recombination, to allow elimination of chromosomal regions carrying deleterious mutations (Kondrashov 1988; Crow 1997).


This work was supported by Medical Research Council grant G9500698MB. We are grateful to the anonymous reviewers of this paper for their comments and acknowledge the secretarial help of Adrienne Knight.

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Genome-Wide Association Study of Base of Tongue Squamous Cell Carcinoma Risk

G.J. Lourenco

B. Carvalho

R. Pellegrino

C.T. Chone

F.F. Costa

C.S. Passos Lima

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Open ArchiveDOI:https://doi.org/10.1016/S0923-7534(20)33587-0

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Inherited genetic alterations, such as single nucleotide polymorphisms (SNPs), were described in association with oropharyngeal cancer risk. However, existing studies have analyzed a limited number of genetic variants. Base of tongue (BT) squamous cell carcinoma (SCC) is a common tumor of oropharynx; however, the association of SNPs and BTSCC risk is still not clarified and, therefore, this was the aim of the present study.


Genomic DNA of 49 BTSCC patients and 49 controls was extracted from peripheral blood samples using the QIamp kit (Qiagen®). Each sample was genotyped individually using DNA high-resolution microarrays containing 500.568 SNPs (SNP array 5.0, Affymetrix®). Further sample processing, including digestion, adaptor ligation, amplification, fragmentation, labeling, hybridization, washing and scanning was assayed according to the standard protocol. Genotype data were acquired by genotyping calling of samples using the crlmm algorithm provided by Bioconductor software. The differences between groups were analyzed by the logistic regression model. The SNPs localized in genes of interest were selected by data base analysis in DAVID and NCBI websites. The validation of selected SNPs was performed by RT-PCR, using TaqMan® SNP Genotyping Assays (Applied Biosystems®) in all samples studied.


We observed 6.609 SNPs with distinct frequencies between BTSCC patients and controls. Fifty-two SNPs (0.8%) were located in coding sequence (CDS), 51 (0.8%) in 3' and 5'-untranslated regions (UTR), 3.461 (52.4%) in up or downstream regions (DWS) and 3.045 (46.0%) in introns. Ten SNPs were selected for validation and eight of them were validated, evidencing those localized in genes related to cell cycle (3'-UTR: ERP29, rs7114; MCC, rs7033; DWS: LEF1, rs2107028 and rs4245926; PTCH1, rs16909856 and rs16909859) and transcription process (CDS: IKBKAP, rs3204145; 3'-UTR: ZNF415, rs3814).


Our preliminary results suggest that SNPs in genes involved in tumor origin and development may predispose individuals to BTSCC in southeastern Brazil. However, the roles of these SNPs in BTSCC susceptibility should be confirmed by functional protein studies and validated in larger epidemiological studies from distinct parts of the world. Financial support: FAPESP and

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