X-chromosome Inactivation, Epigenetics and the Transcriptome
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.
Controls should fore fill the criteria below

 Inclusion Criteria:

 -  Healthy

 -  Age matched

 Exclusion Criteria:

 -  Any chronic or acute illness thought to influence the outcome measures