Ignite Creation Date:
2024-05-06 @ 12:52 AM
Last Modification Date:
2024-10-26 @ 10:56 AM
Study NCT ID:
NCT01678261
Status:
COMPLETED
Last Update Posted:
2016-05-24
First Post:
2012-08-30
Brief Title:
X-chromosome Inactivation Epigenetics and the Transcriptome
Sponsor:
University of Aarhus
Organization:
University of Aarhus
Study Overview
Official Title:
X-chromosome Inactivation Epigenetics and the Transcriptome
Status:
COMPLETED
Status Verified Date:
2015-06
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief Summary:
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 45X Klinefelter syndrome 47XXY triple X syndrome 47XXX and double Y syndrome 47XYY 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 ones own immune system reacts against ones 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 inactivationsilencing of the X-chromosome is initiated by a gene named Xist-gene the X inactivation specific transcriptThis 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-chromosomeas 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
An Increased risk of diseases where ones own immune system reacts against ones 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 inactivationsilencing of the X-chromosome is initiated by a gene named Xist-gene the X inactivation specific transcriptThis 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-chromosomeas 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
Detailed Description:
The X chromosome is a cornerstone to the pathogenesis of a number of syndromes whereof some are Turner syndrome 45X Klinefelter syndrome 47XXY triple X syndrome 47XXX and double Y syndrome 47XYY 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 womens 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 eg 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 eg 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
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 womens 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 eg 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 eg 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
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
None
Is a FDA Regulated Device?:
None
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None