Aortic Aneurysm Clinical Trial
Official title:
X-chromosome Inactivation, Epigenetics and the Transcriptome
The human genetic material consists of 46 chromosomes of which two are sex chromosomes. The
sex-chromosome from the mother is the X and from the father the Y-chromosome. Hence a male
consist of one Y and one X chromosome and a female of 2 X-chromosomes. Alterations in the
number of sex-chromosomes and in particular the X-chromosome is fundamental to the
development of numerous syndromes such as Turner syndrome (45,X), Klinefelter syndrome
(47,XXY), triple X syndrome (47,XXX) and double Y syndrome (47,XYY). Despite the obvious
association between the X-chromosome and disease only one gene has been shown to be of
significance, namely the short stature homeobox gene (SHOX). Turner syndrome is the most
well characterized and the typical diseases affecting the syndrome are:
- An Increased risk of diseases where one's own immune system reacts against one's own
body (autoimmune diseases) and where the cause of this is not known; For example
diabetes and hypothyroidism.
- Increased risk of abortion and death in uteri
- Underdeveloped ovaries with the inability to produce sex hormones and being infertile.
- Congenital malformations of the major arteries and the heart of unknown origin.
- Alterations in the development of the brain, especially with respect to the social and
cognitive dimensions.
- Increased incidence obesity, hypertension, diabetes and osteoporosis.
In healthy women with to normal X-chromosomes, the one of the X-chromosomes is switched off
(silenced). The X-chromosome which is silenced varies from cell to cell. The silencing is
controlled by a part of the X-chromosome designated XIC (X-inactivation center). The
inactivation/silencing of the X-chromosome is initiated by a gene named Xist-gene (the X
inactivation specific transcript).This gene encodes specific structures so called lincRNAs
(long intervening specific transcripts) which are very similar to our genetic material (DNA)
but which is not coding for proteins. The final result is that women are X-chromosome
mosaics with one X-chromosome from the mother and the other X from the father. However,
numerous genes on the X-chromosome escape this silencing process by an unknown mechanism.
Approximately two third of the genes are silenced, 15 % avoid silencing and 20 percent are
silenced or escape depending on the tissue of origin.
The aforementioned long non-protein-coding parts of our genetic material (LincRNAs) are
abundant and produced in large quantities but their wole as respect to health and disease
need further clarification. Studies indicate that these LincRNAs interact with the protein
coding part of our genetic material modifying which genes are translated into proteins and
which are not. During this re-modelling there is left foot prints on the genetic material
which can indicate if it is a modification that results in silencing or translation of the
gene. It is possible to map these foot prints along the entire X-chromosome using molecular
techniques like ChIP (Chromatin immunoprecipitation) and ChIP-seq (deep sequencing).
The understanding achieved so far as to the interplay between our genetic material and
disease has arisen from genetic syndromes which as the X-chromosome syndromes are relatively
frequent and show clear manifestations of disease giving the researcher a possibility to
identify genetic material linked to the disease. Turner and Klinefelter syndrome are, as the
remaining sex chromosome syndromes, excellent human disease models and can as such help to
elaborate on processes contributing to the development of diseases like diabetes,
hypothyroidism, main artery dilation and ischemic heart disease.
The purpose of the study is to:
1. Define the changes in the non-coding part of the X-chromosome.
2. Identify the transcriptome (non-coding part of the X-chromosome)as respect to the RNA
generated from the X-chromosome.
3. Identify changes in the coding and non-coding parts of the X-chromosome which are
specific in relation to Turner syndrome and which can explain the diseases seen in
Turner syndrome.
4. Study tissue affected by disease in order to look for changes in the X-chromosome with
respect to both the coding and non-coding part of the chromosome.
6. Determine if certain genes escape X-chromosome silencing and to establish if this is
associated with the parent of origin.
The X chromosome is a cornerstone to the pathogenesis of a number of syndromes, whereof some
are Turner syndrome (45,X), Klinefelter syndrome (47,XXY), triple X syndrome (47,XXX) and
double Y syndrome (47,XYY). Despite this importance to clinical disease, only one gene on
the X chromosome has so far been implicated in the wide spectra of phenotypic traits seen in
these and other X-related syndromes. The one known gene is the SHOX (the short stature
homeobox) gene and encodes a transcription factor that has brain natriuretic peptide (BNP)
and fibroblast growth factor receptor gene (FGFR3) as transcriptional targets. It is located
at the pseudoautosomal region of the X and Y chromosomes. This gene has been shown to be
involved in short stature in Turner syndrome, Leri-Weill syndrome and idiopathic short
stature. It also causes the increased stature in Klinefelter syndrome, triple X syndrome and
XYY syndrome.
A number of traits and diseases are seen frequently in X-chromosomal syndromes that cannot
be explained by this SHOX gene. The best characterized of these syndromes is Turner
syndrome, where these phenotype traits can be divided into:
1. Autoimmune predilection, which leads to an increased risk of virtually all autoimmune
diseases of unknown pathogenesis such as diabetes and hypothyroidism.
2. Decreased intrauterine viability. Here haploinsufficiency of X-linked pseudoautosomal
genes operating in the placenta has been suggested to be involved (STS and CSF2RA).
3. Ovarian dysgenesis, leading to ovarian insufficiency and the need for long term sex
hormone replacement therapy.
4. Congenital cardiovascular malformations of unresolved pathogeneses.
5. Brain development, especially social-cognitive development, which is altered in many
cases, often in a more "male-like" direction.
6. Increased prevalence of the metabolic syndrome and osteoporosis. In healthy women's
cells, with two X-chromosomes, random X inactivation takes place (13). The process is
governed by the X inactivation center (XIC) and initiated by Xist that is a gene
encoding a long intervening non-coding RNA (lincRNA). The Xist gene is located close to
the centromere on the long arm of the X chromosome, where from it orchestrates
repressive histone modifications (recruiting PRC2) along the X chromosome leading to
inactivation. In the remaining active X chromosome PRC2 is titrated away by Tsix, which
effectively leaves all females as mosaics for the X chromosome with one of maternal and
one of paternal origin. However, a great number of genes that are spread out on the X
chromosome escape this X-inactivation by unknown mechanisms and dosage compensation
takes place, so that expression between males and females are comparable for many genes
(15, 16). Approximately 65% of genes are fully silenced, while 15% completely escape
X-inactivation, and 20% show variable expression, depending on tissue cell origin (17).
LincRNAs are pervasively transcribed in the genome, although their role in health and
disease is poorly understood. Studies of dosage compensation, imprinting and homeotic gene
expression suggest that lincRNAs function at the interface between DNA and chromatin
remodeling with further involvement in reprogramming of chromatin to promote cancer
metastasis. To date a range of different interactions have been hypothesized for lincRNAs in
transcriptional regulation, and they may function both as intact interacting molecules as
well as Dicer processed molecules that are chopped into small interfering RNAs that degrade
other RNAs.
Chromatin remodeling can be analyzed by the marks left by histones on the DNA strand, which
can be of either permissive or repressive nature, depending on the acetylation or
methylation taking place of the histones. As an example, trimethylation of lysine 4 on
histone H3 (H3K4me3) is enriched at transcriptionally active gene promoters, whereas
trimethylation of H3K9 (H3Kme3) and H3K27 (H3K27me3) are present at gene promoters that are
transcriptionally repressed. By use of chromatin immunoprecipitation coupled with deep
sequencing (chIPseq) one can obtain these marks along the whole X chromosome in one assay.
The epigenetic alterations of histone modifications can be studied by a new methodology,
enabling the use of relatively old pathological specimens. This opens new prospects for
expansion of our knowledge of the role of the X-chromosomal permission and inactivation to
different diseases, where X-chromosomal syndromes may serve as the initial model to
understand such processes that are highly likely to be important to diseases (e.g. diabetes
and hypothyroidism) beyond these syndromes. As another example, congenital malformations of
the heart are frequent in Turner syndrome and often lead to early aortic dilatation and
dissection. In these patients and in controls, we collect paraffin- embedded blocks of
tissue from the aortic wall, which can now be assessed using this frontline methodology with
a potential to identify novel marks on Turner patients DNA compared to the DNA of non-Turner
patients.
Imprinting is another important aspect of sex chromosome action. Imprinting refers to the
process where a gene (or more genes) may be imprinted depending on parental origin. Put
another way, a gene can be "turned on or off" depending on its maternal of paternal origin.
Furthermore, mouse studies show that clusters of genes on the X chromosome are imprinted and
are independent of X chromosome inactivation.
The importance of the biological inheritance is apparent for the major cardiovascular
morbidities affecting the population, where a hereditary trait clearly prevails in certain
families. Despite a promise for targeting the prevention and treatment of cardiovascular
morbidity, the specific parts of the genome that potentially trigger the pathologies largely
remain to be defined, and could bring important knowledge of the pathophysiology.
The major body of knowledge on the implications of genome aberrations originates from
diseases with obvious and severe manifestations resulting from clear modes of transmission
that allow identification of the causative regions of the genome. Such genetic disorders
hold the potential for understanding the role of a specific locus of the genome, if this can
be identified, as large chromosomal regions often are involved. In the case of the X-
chromosomal phenotypes we expect the causative agent to be on the X chromosome, and will use
various novel technologies to identify this agent.
Currently, our limited knowledge of the importance of the X-chromosome to cardiovascular
pathology comes from single-gene disorders, and more non-specific gender differences in
addition to the sex chromosomal anomalies. In contrast, no single-gene disorder on the Y-
chromosome has been established to be related to cardiovascular morbidity.
Appropriate human models for improved understanding of the role played by the sex
chromosomes are available. Here, deviations from normality not only occur at a reasonable
prevalence but also associate with readily identifiable phenotypes and adverse prognosis.
Turner and Klinefelter syndromes constitute such models; females with a reduction in X-
chromosomal material and males with an increase in X-chromosomal material, respectively.
These anomalies of the sex-chromosomes associate with excess morbidity and mortality from
both congenital and acquired cardiovascular as well as diabetes, ovarian insufficiency and
other diseases.
The cardiovascular phenotypes and the expression and activation of genes are investigated in
healthy females and males with a comparison between Turner and Klinefelter syndromes in a
cross-sectional descriptive design. These studies have already been performed and a precise
characterization is established. The hypothesis is that the significance of the X-chromosome
will manifest as altered levels of expression and activation in association with different
cardiovascular phenotypes. Secondarily, basic analogous knowledge is provided of the
Y-chromosome. The project is expected to generate further hypotheses on the role played by
the genome to morbidity in both the population having a normal karyotype as well as in
abnormal karyotypes.
In this project we will provide a unique combination of front line molecular technologies
and well defined patient cohorts. The hypotheses we will test are the following:
1. Non-coding transcripts from the X chromosome play a fundamental role in sex chromosome
abnormalities, and may work through regulation of epigenetic mechanisms and through
mRNA destabilization
2. The regulation of non-coding RNA expression on X-chromosomes is based on epigenetic
mechanisms that lead to different histone marks, and different DNA methylation in e.g.,
Turner and Klinefelter syndrome persons when compared to healthy gender-matched
controls.
3. The gene expression pattern resulting from these mechanisms is different in sex
chromosome abnormalities in comparison with healthy males and females, and this
difference can be studied in diseased tissues from Turner syndrome women and compared
to normal control tissue.
4. It may be possible to identify one or a few driver molecules in diseased tissues from
Turner and Klinefelter syndrome persons, that can be validated in vitro and in vivo and
that may explain the disease processes, giving important pathophysiological
information.
Expected findings. We expect to be able to define the epigenetic changes at the
X-chromosomes at a single base resolution, thus identifying CpG methylation at the DNA
strands as well as permissive and repressive histone marks in histones.
We expect to identify the transcriptome both regarding mRNA and non-coding RNAs (long as
well as microRNAs) for RNAs generated from the X-chromosome.
We expect to be able to provide an Atlas of the epigenetic events specific for Turner
syndromes and the effects of these on the transcriptome.
Using bioinformatic methods this will hopefully lead to identification of novel dysregulated
molecules that may explain various properties of these patients. These molecules will then
be subject to validation in separate patient cohorts using PCR or IHC technology.
In diseased tissue we will study the tissue specific alterations of the epigenome and
transcriptome of the X chromosomes and compare this to normal tissues from the control
samples. We hope this will lead to identification of the drivers of the disease process and
a pathophysiological understanding of the disease process.
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Observational Model: Cohort, Time Perspective: Cross-Sectional
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