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X CHROMOSOME ANEUPLOIDY: A LOOK AT THE EFFECTS OF X INACTIVATION A thesis submitted to the Miami University Honors Program in partial fulfillment of the requirements for University Honors with Distinction by Amy Nicole Sprong Miami University Oxford, Ohio May, 2008 ii ABSTRACT X CHROMOSOME ANEUPLOIDY: A LOOK AT THE EFFECTS OF X INACTIVATION by Amy Nicole Sprong The creation of the Barr body has been understood to account for dosage compensation of the X chromosome between males and females. Various X chromosomal aneuploidies such as Klinefelter syndrome (XXY), Turner syndrome(X0), and triple X syndrome (XXX) do not exhibit phenotypic qualities that would indicate that the extra X chromosomes were successfully condensed into Barr bodies. After reviewing current research, it was found that problems with the X inactivation process due to CTCF proteins and CpG islands can lead to some genes on the inactivated X chromosome remaining active. In addition, the X inactivation process does not occur at the beginning of the embryo development, and some genes on the X chromosome always remain expressed. The incomplete and late X inactivation results in the phenotypical differences between XXY, X0, XXX, and normal XX females and XY males. iii iv X Chromosome Aneuploidy: A Look at the Effects of X Inactivation by Amy Nicole Sprong Approved by: _____________________________, Advisor David Pennock ______________________________, Reader Robert Balfour ______________________________, Reader Susan Hoffman Accepted by: ____________________________, Director University Honors Program v vi Acknowledgements Dr. David Pennock Dr. Susan Hoffman Mr. Robert Balfour vii Table of Contents Introduction Meiosis and Mitosis Overview Barr body formation X Chromosome Syndromes Turner syndrome Klinefelter syndrome Triple X syndrome Problems with X Inactivation Possible mechanisms Incomplete X inactivation Timing of X inactivation XIST gene mutations Tying the syndromes together Immunocompetence Height X chromosome reactivated in oocytes Infertility rates Limitations Further research Conclusion References viii 1 2 3 8 8 12 15 18 19 21 21 23 25 25 27 28 29 30 32 36 37 List of Figures Figure 1. Depiction of XIC 4 Figure 2. Possible methods of nondisjunction and resulting offspring 9 ix List of Tables Table 1. Phenotypical characteristics of three sex chromosome disorders 8 Table 2. Possible gametes produced by sex chromosome disorders and the possible offspring 14 x 1 INTRODUCTION Aneuploidy is characterized by a cell having an abnormal number of chromosomes for that cell. A monosomy (missing a chromosome in one or more pairs of chromosomes) and a trisomy (having an extra chromosome in one or more pairs of chromosomes) are aneuploids for humans that normally have 46 chromosomes, or 23 pairs. Aneuploidy can occur due to nondisjunction in meiosis during gamete production, which generally results in non-mosaic offspring, or aneuploidy can be due to nondisjunction in mitosis in the embryo, which generally results in mosaicism in that embryo. Aneuploidy of chromosomes in humans almost always results in embryonic death. However, examples where fetuses with aneuploid chromosomes survive include trisomy 21, trisomy 13, and aneuploidy of the X chromosome. In particular, the X chromosome appears to be the only chromosome in humans that has a mechanism for dealing with aneuploidy. It is thought that embryos with X aneuploidy can survive because of the dosage compensation system placental mammals developed to produce the same number of X chromosome products between males and females. With this system, all X chromosomes except one are condensed and inactivated. In placental mammals, the inactivated X chromosomes are termed Barr bodies. Throughout this paper, it is important to keep in mind that although there has been significant research on Klinefelter syndrome and Turner syndrome, and the conclusions mentioned in this paper about these two syndromes are most likely accurate for the populations because of the large sample sizes, significantly less research has been done 2 on triple X syndrome, and while the conclusions may be accurate for those studies, the small sample sizes indicate those conclusions may not be accurate for the entire triple X syndrome population. Meiosis and Mitosis Overview Generally speaking, meiosis is the division of a single cell to form haploid gametes. The process of meiosis can be broken down into two distinct phases, called Meiosis I and Meiosis II. During Meiosis I homologous chromosomes are separated. During Meiosis II sister chromatids are separated. This is followed by cytokinesis, resulting in the production of four haploid cells. Within Meiosis I, there are four stages: Prophase I, Metaphase I, Anaphase I, and Telophase I. In Anaphase I, the homologous chromosomes are pulled apart by shortening microtubules, and are pulled to opposite poles of the cell. Nondisjunction may occur at this point if the homologous chromosomes are not properly separated and both chromosomes are pulled to the same side of the cell. Meiosis II also has four stages: Prophase II, Metaphase II, Anaphase II, and Telophase II. In Anaphase II, the sister chromatids are pulled apart by the microtubules and the individual chromosomes move towards opposite ends of the cell. As with Anaphase I, nondisjunction may also occur at this point if the sister chromatids fail to separate and aneuploidy can result. 3 Mitosis, the process by which a cell duplicates itself, has an extremely similar mechanism of division as Meiosis II. Prophase, Metaphase, Anaphase and Telophase all have similar steps to Meiosis II. However, since the cell undergoing mitosis is already part of developing organism, nondisjunction in Anaphase results in mosaicism because it is likely that the nondisjunction did not occur in every cell. Barr body formation As explained in Plath’s review article (Plath et al., 2002), Barr bodies are created in mammals through X inactivation when more than one X chromosome is present in the cell. The inactivation occurs as dosage compensation so that the number of active X chromosomes, and therefore the number of proteins encoded by the chromosome in males and females are the same. The inactivation of one of the two X chromosomes appears to be random at the cellular level. Each cell inactivates one X chromosome randomly, independently of any other cell (Plath et al., 2002). At the organ and tissue level, certain genes may also undergo the process of imprinting on the X chromosome, which skews the inactivation away from being completely random for that organ or tissue. Imprinting results in certain genes on one X chromosome being expressed because of the gene’s parental origin. For example, in the placenta, the genes on the paternal X chromosome are silenced so that only the maternal chromosome genes are expressed. (Plath et al., 2002) 4 On the X chromosome, there is a region called the X inactivation center (XIC) (Ng, 2007). In humans, it lies on Xq13. The XIC contains the gene encoding the X inactivation specific transcript (XIST) RNA (Chadwick, 2003). The active XIST gene is found on the active X chromosome, and once made, the XIST RNA acts on the opposite X chromosome – the soon-to-be-inactivated X chromosome (Wutz, 2007). Another gene, called TSIX, is also important for chromosome silencing. The TSIX gene is located at the same locus as XIST, but is read in the opposite direction (Figure 1). Figure 1. Depiction of the XIC. XIST gene is transcribed 5’ to 3’ while the antisense TSIX is transcribed 3’ to 5’. TSIX codes for RNA that is antisense to the XIST gene, and the TSIX RNA blocks the function of the XIST gene. The TSIX gene negatively regulates the XIST gene, and when activated, blocks the inactivation of the X chromosome by binding to the XIST RNA. (Ng, 2007) The TSIX gene is itself regulated by the X-inactivation intergenic transcription element (XITE) region of the X chromosome (Chow, 2005). Before the choice is made as to which chromosome will be inactivated, all X chromosomes have an active TSIX gene, keeping the XIST gene silent. Once it is determined that an X chromosome is to be inactivated, the TSIX on that gene is down-regulated and XIST transcription is up-regulated (Thorvaldsen, 2006). An activated TSIX gene is found on the active X chromosome, because the RNA encoded by TSIX is found near that 5 chromosome, and binds to the XIST RNA, keeping the XIST RNA from binding to that chromosome and inactivating it (Owaga, 2003). In contrast, only XIST RNA is found on the inactivated X chromosome (Plath et al., 2002). Compared to autosomal chromosomes, the X chromosome has significantly more inverted repeats, retrograde sequences, and long interspersed nuclear elements (LINEs), but fewer short interspersed nuclear elements (SINEs) and coding genes (Korenburg, 1988). It is thought that the abundance of LINEs on the X chromosome has some influence on the inactivation of one X chromosome, because no other chromosome undergoes inactivation, and no other chromosome has as many LINE repeats (Lyon, 2006). LINEs are more concentrated around the XIC than they are anywhere else on the chromosome, and are less concentrated on the short arm of the X chromosome, which is less completely inactivated (Lyon, 2003). While the exact mechanism by which LINEs affect X inactivation is unknown, Lyon presents two hypotheses. One hypothesis is that LINEs help promote the binding of the XIST RNA to the chromatin, which would in turn help stabilize the RNA and promote inactivation. Another hypothesis is that the richness of LINEs on the X chromosome causes the X chromosome to have more heterochromatin than euchromatin, because the LINEs are not part of any genes. More heterochromatin before X inactivation might make it easier to stabilize an inactivated X chromosome. (Lyon, 2006) Hetereochromatin is DNA tightly wound to proteins in order to condense the DNA. Because the proteins wind the DNA so tightly, transcription cannot be completed. Further studies are needed to determine the exact mechanism of the LINE effect, but the higher proportion of LINEs on the X chromosome compared to autosomal 6 chromosomes and the concentration of the LINEs on different areas of the X chromosome support the idea that LINEs are involved in X inactivation (Lyon, 2003). The XIST RNA helps initiate chromosome silencing; however, it does not maintain the silencing (Ng, 2007). During the inactivation of one X chromosome, histone modifications occur. These include hypoacetylation of histone H3, demethylation of lysine 4 on histone H3, methylation of lysine 9, lysine 20, and lysine 27 on Histone H3, and ubiquitnylation of histone H2A. Together, these modifications cause transcriptional silencing. Following the inactivation of the chromosome, more histone modifications such as hypoacetylation of histone H4, methylation of the DNA, and the recruitment of macroH2A occur in order to maintain the silencing. (Chow, 2005) CpG islands on the genes are also involved in maintenance of the silencing by providing sites on the inactivated X chromosome for methylation. (Ke, 2003) It is important to note that as many as twenty-five percent of X-linked genes on the inactivated X chromosomes are expressed after inactivation of the chromosome. (Chow, 2005) This perhaps gives insight into dosage compensation for X chromosome aneuploidy, which will be discussed later. Hypothetically, the creation of the Barr body would eliminate the production of any extra proteins encoded by the X chromosome, so that no matter how many X chromosomes are in a cell, the same amount of X-encoded proteins is made. This elimination of extra proteins by the Barr body can be seen when looking at the normal female (XX) when compared to the normal male (XY). The amount of X-encoded proteins in each is similar. However, if this were completely true, then there would be no 7 developmental differences between someone with only one X compared to someone with three. In fact, there are very distinct differences developmentally between someone with Turner syndrome (X0), Klinefelter syndrome (XXY), and triple X syndrome (XXX) when compared to each other and to a normal male and female. The purpose of this report is to try and determine why such differences occur even though a system for dosage compensation already exists. 8 X CHROMOSOME SYNDROMES Turner Syndrome Turner Syndrome is caused by the absence of a Y or second X chromosome, resulting in the X0 genotype, or monosomy of the X chromosome. The 0 represents a missing sex chromosome, meaning only one sex chromosome instead of the normal two is present. Because only one X chromosome is present, X chromosome inactivation does not occur and a Barr body is not created. Common characteristics associated with the syndrome include a webbed neck (pterygium colli), short stature, gonadal dysgenesis, pubertal delay, and lymphedema of the hands and feet. (See Table 1) Phenotype Secondary Sex Autoimmune Characteristics Disease Klinefelter syndrome Tall/Above Small testicles, Present (XXY) average, or incomplete normal development Triple X syndrome Increased lower Normal female, Present (XXX) limb length, or late normal menstruation onset Turner syndrome (X0) Short/below Gonadal Present, average dysgenesis, late hypothyroidism puberty onset Table 1. Table comparing the phenotypical characteristics of three sex chromosome disorders Klinefelter syndrome, triple X syndrome, and Turner syndrome. Height appears to be correlated with number of X chromosomes. Sex Chromosome Disorder Height/Stature However, many other physical attributes are associated with Turner syndrome. (Doswell, 2006) Psychological problems can also be associated with the syndrome. (Ogata, 1994) 9 However, the majority of females with Turner syndrome do not have psychological problems, and those that do receive inconsistent diagnoses, and thus no single psychiatric problem is synonymous with the syndrome (Catinari, 2006) The X0 genotype can arise from many different complications in meiosis (Figure 2). Figure 2. Possible methods of nondisjunction and resulting offspring. Embryonic lethals Y0, YY, and 00 were not included, and mitotic nondisjunction was not considered. KF is Klinefelter syndrome, TS is Turner syndrome, and TX is triple X syndrome. The XYY “super male” is seen with the aneuploidy of the Y chromosome and is not included in this essay. 10 Normally, one parent each provides a sex chromosome. However, if during meiosis the gametes undergo nondisjunction, an egg or sperm in the final stages of production can be left without a sex chromosome, and the other left with two X chromosomes in an egg, or an XY, XX, or YY in a sperm, depending on during which stage of meiosis nondisjunction occurs. If the gamete without a sex chromosome joins with a gamete from the opposite sex with a normal X chromosome, the resulting zygote is X0, or the Turner syndrome genotype. The majority of the X chromosomes in women with Turner Syndrome are maternal in origin, meaning the X chromosome came from the mother and the father donated no sex chromosome. (Frias, 2003) The reason for the generally maternal origin of the X chromosome will be discussed later. While females with Turner Syndrome are generally infertile, it is possible for the production of viable gametes to occur. If this does occur, the female theoretically has a 50% chance of producing an X carrying egg, and a 50% chance of an egg without a sex chromosome because of her X0 genotype. If the blank egg combines with an X carrying sperm, then an X0 zygote is once again created. The single copy of the X chromosome in patients with Turner syndrome results in haploinsufficiency for the X chromosome, similar to males (Doswell, 2006). Haploinsufficiency causes any recessive traits on the remaining X chromosome to be expressed, because there is no second X chromosome to carry the dominant allele to “cover” the expression of the recessive. This may be one of the causes of the developmental difficulties of women with Turner Syndrome, because many harmful 11 developmental traits are carried recessively on the X chromosome, such as hemophilia. (Frias, 2003) Therefore, females with Turner Syndrome are more likely to express hemophilia than those without Turner Syndrome, much like males, because if the female has Turner syndrome, they only need to have one copy of the recessive gene to exhibit hemophilia, whereas a female without Turner Syndrome would need to have two copies of the recessive gene, which is much rarer. Most X0 females spontaneously abort in the womb, while the majority of known living Turner cases are mosaics (Frias, 2003). Mosaicism occurs when there are two or more cell lines in an individual that are chromosomally different (Frias, 2003). Mosaicism in Turner Syndrome is due to mitotic nondisjunction. This could happen if one cell of an XX embryo underwent mitotic nondisjunction, which would result in a daughter cell of X0 and another other daughter cell of XXX. The resulting genotype would be X0/XX/XXX. (Antich, 1967) The fact that the majority of living Turner syndrome cases are mosaics suggests that the additional X chromosome contains something necessary for survival (Frias, 2003). Girls who are mosaics are more likely to experience more normal developmental patterns such as growth spurts, spontaneous menstruation, and secondary growth characteristics (Doswell, 2006). The somewhat normal developmental patterns in mosaics presumably occur due to the presence of the second X chromosome in certain cells of the body. 12 Klinefelter Syndrome Klinefelter Syndrome is characterized by the presence of extra X chromosomes in males, resulting in genotypes such as XXY, XXXY, and XXXXY. The occurrence of Klinefelter syndrome in the population is between 0.1% and 0.2% (Lanfranco, 2004). The probability of an individual having the genotype and surviving decreases as the number of X chromosomes increases, since the developmental defects become more severe. The number of Barr bodies created is dependent on the number of X chromosomes in the individual. For an XXY male, X inactivation will randomly occur in one of the X chromosomes in each cell, resulting in one Barr body. The most common characteristics associated with Klinefelter syndrome are small testicles and incomplete development of secondary sex characteristics. All other characteristics, such as sparse body hair, gynecomastia, and body size are variable. (Visootsak, 2006) These characteristics are often described as ‘feminized’ male characteristics, which are due to the extra X chromosome (Milunski, 2001). Testosterone levels are generally low and gonadatrophin levels are high, and there is incomplete development of secondary sex characteristics due to a decrease in androgen production (Visootsak, 2006). Besides physical problems, psychological problems may also be associated with Klinefelter syndrome (Ogata, 1994). While the psychological impact of the syndrome is generally low and a very few patients have mild to severe retardation, psychological problems associated with Klinefelter syndrome in the past include language-based learning difficulties, executive dysfunction, and anxiety due to the underdeveloped 13 secondary sex characteristics (Geschwind, 2004). Despite these characteristics, as many as 60% of Klinefelter cases are suspected to be undiagnosed due to minimal expression of the symptoms of the disease (Wikstrom, 2006). The XXY genotype can arise from different complications in mitosis and meiosis (See Figure 2). In meiosis, one gamete may undergo nondisjunction, resulting in two different gametes; one with two sex chromosomes and the other with none. (Wikstrom, 2006) In the egg, that would leave a gamete with XX. If the XX egg were to be fertilized by a Y carrying sperm, the resulting zygote is XXY. Nondisjunction may also happen in the sperm, resulting in a sperm not carrying a sex chromosome, and a sperm carrying XY. If the XY sperm then fertilizes an X carrying egg, the XXY zygote is created. XXXY may occur when both gametes undergo nondisjunction. Nondisjunction during mitosis once the zygote has already been created would lead to a mosaic form of Klinefelter Syndrome; however, only 15% of males with Klinefelter syndrome are mosaics (Mark, 1999). Klinefelter males are also generally infertile, but some can still have children (Ogata, 1994). Theoretically, Klinefelter males have the possibility of producing X, Y, XX, and XY gametes. (See Table 2) 14 Possible Gametes Produced Resulting offspring phenotype Normal female Klinefelter Normal male syndrome Triple X female (XXY) Klinefelter male Turner Syndrome Normal male or (X0) female 0 X0 or Y0 Turner female* or embryonic death Triple X syndrome XX XXX or XXY Triple X female (XXX) or Klinefelter male X XX or XY Normal male or female Table 2. Table showing all possible gametes produced by the sex chromosome disorders discussed in this text and their possible offspring if they mated with a normal diploid partner. *The likelihood of this female offspring being born is very low due to the blank chromosome being expressed in the placenta, and will most likely spontaneously abort. Sex Chromosome Syndrome X Y XX XY X Resulting offspring genotype with normal genotype partner XX XY XXX XXY XX or XY If these gametes were fertilized, the offspring has the possibility of have a normal male or female genotype, or a Klinefelter or triple X genotype. Genes on the X chromosome are also involved in making receptors. One receptor of particular importance is the androgen receptor. It is thought that the receptor gene might undergo some slight changes in Klinefelter syndrome, such as adding more CAG repeats (CAGn) (Wikstrom, 2006). “In Klinefelter patients who have two androgen receptor alleles [due to the presence of two X chromosomes], the shorter CAGn allele is preferentially inactive. CAGn length is positively associated with body height. Bone density and the relation of arm span to body height are inversely related to CAGn length. Presence of long CAGn is predictive for gynecomastia and smaller testes (Zitzmann, 2005).” More CAG repeats appear to lower the affinity of androgens to the receptor, although the exact mechanism by which this occurs is unknown. This combined with the already low 15 levels of androgens such as testosterone may lead to some of the secondary sex characteristics associated with the disease. (Wikstrom, 2006) Instead of random inactivation, the gene inactivation is skewed, and the genes with longer CAGn are generally active (Zitzmann, 2004). Triple X Syndrome Triple X Syndrome occurs in females who have more than the normal two X chromosomes. Triple X syndrome is not confined to the XXX genotype. It also includes the addition of more than one extra X chromosome. However, as with Klinefelter Syndrome, the likelihood of survival decreases with the addition of each X chromosome, so XXXX and XXXXX are extremely rare. While there are generally more psychological problems than physical, common characteristics include an increased lower limb length, developmental difficulties in offspring of affected females, and immune difficulties because of increased serum IgM levels. (Goswami, 2003) These characteristics can be variable, however, and any one case would not necessarily have all of the characteristics. While a few characteristics associated with the syndrome have been mentioned, a specific phenotype for this syndrome does not exist (Tennes, 1975). (See Table 1) This is due to the extreme variability that is seen in each of these characteristics between different cases of females with Triple X syndrome and the small number of documented cases. As with Klinefelter, it is estimated that many triple X cases are unreported because they experience mild, if any, characteristics of the syndrome. (Milunski, 2001) 16 Similarly to Turner Syndrome and Klinefelter Syndrome, the triple X genotype of XXX may be created through nondisjunction of the gamete chromosomes during meiosis, or nondisjunction of the chromosomes of the zygote during mitosis (See Figure 2). If one gamete undergoes nondisjunction during meiosis, one gamete with two of the chromosomes that underwent nondisjunction and one gamete with none of that particular chromosome are created. If, for example, the egg is the gamete that underwent nondisjunction of the sex chromosomes, an XX egg would result. If the XX carrying egg is fertilized by an X carrying sperm, the genotype XXX would result in the zygote. A normal XX zygote could also undergo nondisjunction of the sex chromosomes during mitosis resulting in numerous different outcomes, each depending on when in the developmental process they occurred. One possible outcome is the creation of a mosaic XXX,X0. An XXX,XX,X0 mosaic case has also been reported (Chen, 2003). Triple X females, like the other syndromes, are generally infertile but have been known to have children (Goswami, 2003). Assuming nondisjunction did not occur in the triple X female’s gametes, they have the possibility of producing X or XX eggs. (See Table 2) If the eggs are fertilized the resulting embryo could be a normal male or female genotype, or a Klinefelter or triple X genotype. (See Table 2) Developmental difficulties in the offspring of females with triple X syndrome involve cardiac, neural tube, and genitourinary tract defects. It is thought that the mother’s extra X chromosome causes a higher than normal amount of X-encoded proteins to be in the womb, which act negatively on the embryo’s development. These 17 defects generally cause the children of a female with triple X syndrome to die during childhood, thus increasing offspring mortality rates for the syndrome. (Goswami, 2003) 18 PROBLEMS WITH X INACTIVATION It has been found that at least 30 X-linked genes are expressed on the inactivated X chromosome. (Basrur, 2004) However, the number of expressed genes on the inactivated X chromosome is also estimated at up to 25 percent of genes on the X chromosome. (Ke, 2003; Chow, 2005) The X chromosome contains between 900 to 1400 genes of the estimated 20,000 to 25,000 genes in the human genome (NLM, 2006).The disagreement on the number of genes that escape X inactivation is because different research projects have yielded different numbers, and some genes on the inactivated X chromosome have been found to escape X inactivation only part of the time (Ke, 2003). This means that even though the majority of genes on one of the X chromosomes are inactivated and most genes of both the X chromosomes in a normal diploid female are only being expressed by one chromosome, certain genes are being expressed twofold. While this could potentially create a difference between male and female expression levels, many of these genes are found to have a homolog on the Y chromosome that is also being expressed. (Disteche, 1997) The expression of these 30 or more genes on the inactivated X chromosome may be one reason for the developmental differences in Turner, Klinefelter, and triple X patients. In Turner Syndrome, the 30 genes are only being expressed at half of what they are in diploid males and females due to the missing second sex chromosome. In both Klinefelter Syndrome and triple X syndrome, the third sex chromosome would lead to those 30 or more genes being expressed one-and-a-half fold what they would be in a diploid male or female. 19 Possible mechanisms Genes on the inactivated X chromosome that escape inactivation often have homologs on the Y chromosome (Disteche, 1997). The Y homologs often have a percentage of different nucleotide sequences relative to the corresponding X chromosome genes, suggesting a change of function over evolutionary time (Disteche, 2002). The Y homologs can code for proteins or for nothing at all, and some promote the transcription of genes on the X chromosome. (Disteche, 1997) CpG islands are cytosine and guanine bases separated by a phosphate on the DNA strand. CpG islands at the 5’end of the genes that escape X inactivation are not methylated, causing the inactivation process to ‘skip’ over that particular gene or area (Disteche, 2002). In order for a gene to be initially silenced, the CpG island needs to be methylated. Areas that are low in CpG islands are more likely to remain activate after the rest of the chromosome has been inactivated because the DNA cannot be methylated, which would allow inactivation (Ke, 2003). However, while CpG islands and their methylation are important for the initiation of silencing, they are not essential for maintaining the silencing (Filippova, 2005). A study on one of the few genes that escapes X inactivation in the mouse, Scmx (also known as Jarid1c) found a bound protein that appears to be at least partially responsible for the escape. The bound protein, CCTC-binding factor (CTCF), is a chromatin insulating factor found on the 5’ end of the escaped gene, at the boundary between it and an inactivated gene. Previously, CTCF had been found to be important in 20 the maintenance of preventing methylation. (Filippova, 2005) This function is especially important because DNA must be methylated in order to be condensed, and condensing a gene causes inactivation (Ke, 2003). Therefore, CTCF may help genes to escape inactivation by preventing methylation at the start of the gene. CTCF is only involved during the maintenance of escaping inactivation, and is not involved in the initiation of inactivation. Initially, the gene may be inactivated with the rest of the inactivated X chromosome if it has, for example, a CpG island. However, when the DNA methylation occurs on the chromosome in order to propagate maintenance of the inactivation, the genes that have bound CTCF would not be methylated. The genes with CTCF would therefore be protected from methylation and would be reactivated. (Filippova, 2005) The CTCF gene is also found in humans in addition to mice, and more importantly, it is found on the inactive X chromosome (Xie, 2007). CTCF is thought to interact with the XIST gene’s promoter in addition to preventing methylation of genes that escape X inactivation (Pugacheva, 2005). Mice allow fewer genes on the X chromosome to escape inactivation than humans. They also show less severe phenotypic effects from aneuploidy of the X chromosome than humans do (Disteche, 1997). Assuming that these observations are correct, then it may be concluded that humans suffer greater phenotypic effects, such as infertility, from X chromosomal aneuploidy due to our inability to inactivate the entire X chromosome. In addition, it can be concluded that the genes which escape X inactivation on the inactivated X chromosome are the real cause of the developmental differences in humans with Turner, Klinefelter, and triple X syndrome (Disteche, 1997). 21 Incomplete X inactivation The genes that escape X inactivation are expressed at a lower activity level than the same genes on the active X chromosome (Disteche, 1997). This could be due to the position of a gene on the inactivated chromosome in close proximity to heterochromatin (Disteche, 2002). If genes are located close to heterochromatin, it may be more difficult for transcription factors to bind and begin transcription, resulting in a lower expression of the gene. Genes that escape inactivation are expressed to varying degrees, depending on the tissue and the individual. (Disteche, 2002) These varying degrees of expression may result in the varying phenotypes seen within each syndrome, such as why some females with triple X syndrome experience mental retardation, but others do not. Timing of X inactivation X chromosome inactivation occurs during the late blastula/early gastrula stage of the embryo. (Migeon, 2007) Therefore, in diploid females, Klinefelter males, and triple X females, all of the X chromosomes in cells are active before the gastrula stage. It has been determined that genes on the X chromosome are expressed during that time, which is shown by a bimodal distribution of the gene protein dosage during one study (Epstein, 1978), with the lower mode representing the male dosage, and the higher mode representing the female dosage. In addition, the higher mode was two times the amount 22 of the lower mode, which makes sense because females have two X chromosome compared to males who have one (Epstein, 1978). The Epstein study only focused on one gene on the X chromosome, HGPRT (Epstein, 1978). Based on the twofold difference in amount of the protein between sexes, it is not a gene that is expressed or has a homolog on the Y chromosome in males, but the study still shows that all X chromosomes remain active during a period of time in development. Other genes on the X chromosome, however, have a functional counterpart on the Y chromosome (Disteche, 1997). While the HGPRT gene and its corresponding protein may not be essential in specific amounts and may vary based on the number of X chromosomes (as in males and females), other proteins may have dosage-dependent effects and changes in these levels would affect developmental patterns (Epstein, 1978). Patients with Klinefelter syndrome or triple X syndrome have an extra chromosome contributing genes for a certain period of time during development. In females with Turner syndrome, only one X chromosome is active because only one exists. If the hypothesis that all of the X chromosomes are active during early development and that they affect development at that time is true, then for a cell to develop according to what we would define as a normal female standard, two X chromosomes would need to be present until the late blastula stage. Klinefelter males have this; however, they also have a Y chromosome that is contributing genes and proteins (although the Y has only 1/10 of the number of genes of the X chromosome). It is possible that both X chromosomes together cause part of the reduction of male characteristics, such as testicular formation and size, since two X chromosomes are 23 generally only present in females. Further research would need to be conducted in order to determine if this is a feasible explanation for the Klinefelter phenotype. The dosage of X chromosomes is also important for the development of the Turner syndrome phenotype. Turner syndrome females would lack those extra chromosomal genes from the second X chromosome during the first few stages of development. This would result in an altered dosage of the proteins for a short period of time, leading to the development of the phenotypic characteristics of Turner syndrome. (Ogata, 1994) XIST gene mutations XIST gene mutations, depending on the specific defect, can result in normal X chromosome inactivation, partial X inactivation, or no X inactivation at all. A mutated XIST gene is often seen on the inactivated X chromosome, because while the XIST RNA is made on one X chromosome, it acts on the opposite X chromosome. A mutated XIST gene is less likely to encode for an XIST RNA that can inactivate the other X chromosome, and the normal XIST gene is more likely to inactivate the X chromosome with the mutated XIST gene, (Migeon, 1998) One of the most common mutations on the XIST gene is in its CTCF region. An XIST gene mutation in the CTCF region may affect the binding of CTCF during inactivation, depending on the type and severity of the mutation. C-T-C is the normal nucleotide sequence in the CTCF region, but a mutation that changed the sequence to CG-C increases the binding of CTCF. A mutation in the sequence changing it to C-A-C 24 decreases the binding. (Pugacheva, 2005) While the C-G-C mutation would cause more genes to escape X inactivation because of the increased binding of CTCF, the C-A-C mutation would cause fewer to escape. Both of these mutations could contribute to the variability we see in patients with aneuploidy. Causing fewer genes to escape X inactivation could reduce the dosage of X-linked genes and the proteins they encode in triple X syndrome and Klinefelter syndrome down to more normal levels, resulting in a less severe phenotype. However, causing more genes to escape X inactivation may lead to some of the more severe phenotypes due to gene dosage problems. 25 TYING THE SYNDROMES TOGETHER Studies have found that the X chromosome is responsible for genes expressed in muscle, germ, and brain cells. (Ng, 2007) Therefore, an extra or missing X could affect the way these areas develop. The inability of the person affected with triple X syndrome to have children that live past early childhood is an example where the extra X chromosome genes affect germ line and placental cells. Immunocompetence The X chromosome is thought to carry genes that are involved with immunocompetence. One reason for this thought is the prevalence of autoimmune thyroid disorder among females with triple X syndrome and in patients with Turner and Klinefelter syndrome (See Table 1) (Goswami, 2003; Brix, 2005). The Y chromosome does not have homologs to the immunocompetence genes on the X chromosome, but the Y chromosome does have a gene involved in autoimmunity (Invernizzi, 2005). Females with Turner syndrome make up a significant proportion of people with autoimmune thyroid disease. (Invernizzi, 2005) In Turner females, the prevalence of autoimmune thyroid disorder is due to the hemizygosity of the X chromosome. Immunocompetence genes escape X inactivation, meaning the normal dosage for the genes comes from two X chromosomes, and patients with Turner syndrome receive half of the normal dosage. (Miozzo, 2007) One hypothesis is that some genes that escape X 26 inactivation play a role in immunocompetence susceptibility, and the Y chromosome has genes that create autoimmunity. The idea that the Y chromosome has genes that affect autoimmunity is supported by the higher incidence of females who have an autoimmune disease when compared to males. (Invernizzi, 2005) This hypothesis, however, fails to explain why males with Klinefelter syndrome (who have a Y chromosome) still express high rates of autoimmune diseases. Estrogens (from the X chromosome) may exaggerate autoimmune disease while androgens (from the Y chromosome) may inhibit it, which may explain why the Y chromosome may cause a lower incidence of autoimmune diseases in males (Brix, 2005; Oktenli, 2002). In addition, the extra X chromosome in Klinefelter males is producing more estrogen than a normal male would have, which could explain why Klinefelter males have a higher incidence of autoimmune disease than XY males. Another hypothesis is that the Y chromosome in males plays a protective role against immune diseases. (Invernizzi, 2005) The second hypothesis, however, only explains why autoimmune diseases are seen in higher numbers in patients with Turner syndrome, and does not explain their frequency with Klinefelter syndrome or triple X syndrome. Autoimmune liver disease, another immunocompetence disease, is also seen with increased rates in females with Turner syndrome. This is also thought to be due to the hemizygous nature of their X chromosome. (Invernizzi, 2005) The correlation between autoimmune diseases and these syndromes gives support to the idea that at least one gene involved with the immune system and its maintenance is 27 located on the X chromosome, and aneuploidy of the chromosome alters the dosage effect. Height As noted before, Turner syndrome results in haploinsufficiency of the X chromosome because there is only one copy of the chromosome. The short stature homeobox containing (SHOX) gene is located on the short arm of the X chromosome, and when subjected to haploinsufficiency, results in short stature. This is evident in females with Turner syndrome, who fall in the lowest range of average human height. (Kosho, 1999) The SHOX gene is also one of the more than 30 genes on the inactivated X chromosome that escapes inactivation, so dosage is altered with X chromosomal aneuploidy. (Blaschke, 2006) In addition, the SHOX gene is also found on the Y chromosome (Ogata, 2001). A common characteristic of triple X syndrome is increased lower limb length, which could add to an overall height increase. One possible explanation for the increase in height compared to a normal female is the SHOX gene is expressed on three X chromosomes, resulting in a higher than normal dosage. (Kanaka-Gantenbein, 2004) The SHOX gene appears to have additive characteristics (Ogata, 2001). The greater the number of copies of the SHOX gene, the taller the overall height of the person. The reverse is also true; as the number of SHOX alleles decreases, so does height. If a normal dosage affecting height is due to two alleles, one on each sex chromosome, 28 then patients with Turner syndrome would be shorter than normal due to only having one copy of the X chromosome, and triple X patients would be taller than normal, due to having three copies of sex chromosomes, and a general overdosage of the gene. Males with Klinefelter also tend to be taller than normal. While they do grow at a normal rate through puberty timelines, males with Klinefelter syndrome continue growing past the timeframe when normal diploid males would stop. (Ogata, 2001)This is most likely due to having three copies of sex chromosomes. X chromosome reactivated in oocytes X chromosome inactivation is reversed in germ line cells of the body, so a normal diploid female would have one inactivated X chromosome and one activated X chromosome in their somatic cells, and two activated X chromosomes in their oocytes. However, a female with Turner syndrome would only have one active X chromosome in her oocytes, and a female with triple X syndrome would have three active X chromosomes. This would result in decreases and increases of X chromosome product dosages for the oocyte during meiosis in females with Turner and triple X syndromes, respectively. These changes in protein dosage may then lead to the gonadal dysgenesis related to the syndromes, and cause high infertility rates. (Ogata, 1994) The high infertility rates would be the result of the fewer or extra copies of proteins, depending on the syndrome, acting on the developing embryo and the uterus. An imbalance may cause abnormal development, at which point the embryo may spontaneously abort. 29 Infertility rates Another cause for spontaneous abortion in females with triple X syndrome may be due to the organization of genes in the placenta. In the placenta, X inactivation occurs for dosage compensation; however, it is not random. Instead, the paternal genes are inactivated, and imprinting occurs. (Reik, 2005) Therefore, if the mother is XXX, and she donated an XX genotype to her baby (making the baby an XXX, or XXY; refer to Table 2), two X chromosomes remain activated in the placenta, and dosage compensation does not occur as it would in a normal female. As mentioned earlier, the majority of the X chromosomes in females with Turner syndrome are maternal in origin, meaning the father had an empty gamete and did not donate a sex chromosome (Milunski, 2001). If this information is compared to what genes remain active in the placenta due to imprinting, the reason for the paternal origin of the missing X chromosome is easy to see. If the mother were to not contribute a chromosome, when imprinting occurred in the placenta, the paternal chromosomes would be inactivated and there would be no X chromosome to create proteins, resulting in embryonic death. Therefore, the mother must donate the X chromosome, and because the blank chromosome from the father would have been ignored through imprinting anyway, the baby is carried to term. 30 LIMITATIONS Unfortunately, there is very limited research on triple X syndrome compared to Turner and Klinefelter syndromes. This is most likely because a large majority of triple X cases go unreported, since there is no distinct phenotype. The vast majority of reports on the syndrome involve only one to five subjects, which is an extremely small number to take common characteristics from and then apply it to the entire population of females with triple X syndrome. In addition to the small number, most of the case studies are not on females afflicted with just triple X syndrome, because the females are often seeking treatment for some other disorder when their triple X genotype is discovered. Instead, the patients have at least one other disorder, whether it be another genetic disorder such as Trisomy 21, or a rare developmental disorder. This unfortunately skews the results, and characteristics from these patients may not be entirely due to triple X syndrome and may instead be due to another affliction or the combination of the two disorders. The research on autoimmune disorders is also limited. Only 2 reports on triple X syndrome and autoimmunity exist. (Goswami, 2003) Even if the two cases were combined, the numbers are still statistically too small to draw any type of definite conclusion. As mentioned in the synopsis of Klinefelter, Turner, and triple X syndrome, mosaics can exist in many different combinations. However, many reports do not mention testing for mosaicism in the subjects. It is unknown if all of the cells in the subjects had the same genotype, such as being purely X0, or purely XXY, or if there was 31 a mix of mosaics and non-mosaics included in the population for the syndrome. This is important because non-mosaics are more likely to have a more severe phenotype of the syndrome as compared to mosaics, because mosaics may still have some cells that have the normal XX or XY chromosomes to offset the effects of the aneuploidy cells. Failing to test for or acknowledge these differences skews the results. It was noted throughout the paper that a varying number of genes that escape inactivation on the inactivated X chromosome were presented. This is due to conflicting reports in the literature that estimate the number from as low as 30, to as high as 25 percent of the thousands of genes on the X chromosome. While this may be due to different time periods when the reports were written and to which genes had been discovered to escape inactivation, it is also due to how the writer defined “escaping” from X inactivation. There are three classes of genes on the X chromosome in terms of inactivation. There are those that are always inactivated, those that are always active (a small number), and those that can be activated or inactivated (generally a fairly large number) depending on the person. Some of the researchers included the always and sometimes active groups, while others only counted the always active genes as those that escape X inactivation. LINEs were mentioned to have an influence on X chromosome inactivation. While this explains why the X chromosome can be inactivated compared to the autosomal chromosomes, it does not explain why the inactivation is random and does not occur to all of the X chromosomes. 32 FURTHER RESEARCH As mentioned in the limitations section, the small number of triple X syndrome cases creates a difficulty in studying the syndrome. The triple X cases that also have a second disorder may be because many triple X syndrome cases go unreported due to the lack of a distinct phenotype, as mentioned earlier. A few of the case studies mentioned finding the triple X syndrome females by accident; the researchers were actually trying to look at another genetic anomaly, such as fragile X syndrome, and came across the triple X females. In order to try and create the possibility for more studies on triple X syndrome females unafflicted with an unrelated disorder or another genetic disorder, it may help to screen all newborns for these genetic syndromes. This would in turn inform the patient that they have a syndrome, even if they exhibit no characteristics, and then they may make the choice to participate in a future study. However, the possibility of this occurring is an ethical dilemma that cannot be adequately addressed in this paper. Another idea for future research is to look at the Barr body formation mechanism. While it is already suspected the XIST RNA, CpG islands, and CTCF are involved in Barr body formation in one way or another (whether it be to help create the Barr body, or to block condensation of a specific gene or the entire chromosome), the exact role may be further determined by blocking these different proteins by themselves or in a group. It can then be determined if, for example, CpG islands are essential for creation of the Barr body, or if the XIST RNA alone may create the X chromosome inactivation. Also, by blocking CTCF, one could see if a gene escaped X inactivation a certain percentage of 33 the time, indicating that CTCF is not the only protein involved in blocking gene inactivation. In addition, experiments which block certain parts of the Barr body creation mechanism may uncover more specific aspects and functions of process by allowing the cells to divide and develop into an organism, then looking at possible incomplete Barr body development and its effects on the resulting embryo. It is known that both the X chromosomes are activated and express the genes located on them before Barr body condensation occurs. However, the specific effect this activation has on the cell is unknown. An idea for a future research experiment would be to try and inactivate one X chromosome in a normal diploid female embryo during the time that both Xs would normally be active. Once the embryo passes to the early gastrula stage, it should behave like a normal embryo because one X chromosome is already condensed, and the condensed chromosome can be allowed to express whichever genes it normally would during X chromosome inactivation. Whatever developmental differences occur when compared to control embryos at the same stage (preferably a clone to the original cell used) would be due to the lack of both X chromosomes being expressed during early development. In addition, the differing characteristics, if there are any, would most likely resemble the phenotypic characteristics of Turner syndrome (out of Turner syndrome, Klinefelter syndrome, and triple X syndrome) due to the similarity of chromosomal composition during those early stages before Barr body condensation. Mice allow fewer genes be expressed on the inactive X chromosome, and it is assumed the smaller number of genes that escape X inactivation is the reason they express lesser X0, XXX, and XXY phenotypes. This assumption could be tested by 34 creating a way to fully block the extra sex chromosome in XXY or XXX genotypes in an organism that expresses the syndrome’s corresponding phenotypes readily, and looking at the resulting phenotypes after blocking the extra sex chromosome. Fully blocking the extra chromosome, including the genes that normally escape X inactivation, would eliminate the extra proteins created by expressed genes on the third chromosome. A similar experiment would be to add the proteins that would normally be created on an inactivated X chromosome into an organism with the genotype X0, and then looking at the phenotypic effects. This is not likely to be an easy task, but if the mechanism were created to completely inactivate an extra X chromosome, or add the products from a missing X chromosome, then if caught early enough, the phenotypic effects of the genetic syndromes discussed in this paper could be essentially reversed. This is, however, a daunting task because all of the genes that escape X inactivation have still not been identified, and we still have no definite mechanism for determining why one gene escapes X inactivation some of the time but not all of the time. Further studies on why that occurs would also be needed. Disetche mentioned that the proximity of a gene to heterochromatin may be related to the gene’s expression level (Disetche, 2002). This could be tested by selecting varying expressed genes on the inactivated X chromosome and comparing their distance from the heterochromatin to the gene, and the gene’s average level of expressivity across varying subjects (i.e., testing for Scmx expressivity and the distance from heterochromatin in different people). If Disetche is correct, then we would see higher expressivity the further the gene is from the heterochromatin. 35 The presence of two X chromosomes in a male, as in Klinefelter syndrome, appears to cause less masculine characteristics. The mechanism for this is unclear, however. An interesting continuation of that research would be to try and determine if the extra X chromosome only dilutes the expression of the Y chromosome, or if the extra X creates competition for the development of the secondary sex characteristics. The second statement is plausible due to the female sex being the “default” gender, while the male state is the alternative due to the SRY gene. If the default is producing more female proteins than usual, it may be more difficult for the male gender to be assigned based on the SRY gene alone. Another idea for future research would be to look into autoimmune diseases and Klinefelter syndrome. If autoimmune diseases are caused by low levels of testosterone, then a study looking into using androgen replacement therapy on males with Klinefelter syndrome may reduce the incidence of autoimmunity related diseases. In addition, if the replacement therapy is started early enough, another phenotype of Klinefelter syndrome, the immature or smaller secondary sex characteristics, may be returned to normal ranges. 36 CONCLUSIONS Further research still needs to be conducted, but based on the findings until now, incomplete and late X inactivation of extra X chromosomes appears to be the causes of the phenotypical differences between Turner syndrome, Klinefelter syndrome, triple X syndrome, and XY male and XX female. While the genes that escape X inactivation are important for development, when the escaped genes aren’t there because there is no extra X chromosome (as in Turner syndrome), or when there are too many copies of the escaped genes because there are too many extra sex chromosomes (as in Klinefelter syndrome and triple X syndrome), developmental difficulties can occur. The varying degree of phenotypes within each syndrome occurs because the genes that escape X inactivation are expressed at varying degrees depending on the person, and the number and type of genes that escape X inactivation can vary from person to person. The major limitations of this paper include limited research on triple X syndrome and the possible inclusion of mosaics compared with non-mosaics due to authors failing to make that distinction in the research studies. 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