Download X Chromosome Aneuploidy: A Look at the Effects of X Inactivation

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
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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.
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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
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Acknowledgements
Dr. David Pennock
Dr. Susan Hoffman
Mr. Robert Balfour
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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
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List of Figures
Figure 1. Depiction of XIC
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Figure 2. Possible methods of nondisjunction and resulting offspring
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List of Tables
Table 1. Phenotypical characteristics of three sex chromosome disorders
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Table 2. Possible gametes produced by sex chromosome disorders and
the possible offspring
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x
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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
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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.
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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)
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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
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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
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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
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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.
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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)
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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.
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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
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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.
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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
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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)
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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
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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)
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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
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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)
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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.
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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
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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).
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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. Further research ideas include more general research
on triple X syndrome, comparing gene expressivity to distance from heterochromatin,
and further studies on autoimmune diseases in all three syndromes mentioned in this
paper.
37
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