Classic Bladder Exstrophy: identification of genetic markers and characterization of its associated ISL1 gene
Classic Bladder Exstrophy: identification of genetic markers and characterization of its associated ISL1 gene
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DissertationPrüfungsdatum
17.10.2023Datum der Veröffentlichung
24.09.2024Erstgutachter
Odermatt, BenjaminZweitgutachter
Pankratz, MichaelMetadaten
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Inhalt
Classic bladder exstrophy (CBE) is a congenital anomaly with an occurrence of 1 in 30.000 new born. It is characterized by the protrusion of the bladder plate through an open abdominal wall, often accompanied by kidney and upper urinary tract abnormalities. Its long-term complications include different bladder malignancies.
While the phenotype of CBE is well understood, the genetic and molecular causes remain largely unknown. Previous genetic studies have identified one locus associated with CBE, but these studies had limited sample sizes. The aim of this study is to identify new risk loci and gain a better understanding of their contributions to CBE and CBE-associated cancer. Furthermore, the study aims to elucidate the molecular mechanisms through which the associated ISL1 gene contributes to CBE.
For that, this study shows here the largest genome-wide association study (GWAS) conducted on CBE to date, identifying eight genome-wide significant loci, seven of which are novel. Within these loci, ten coding and four non-coding genes are found. RNA-seq analysis of mouse and human embryonic bladder tissues at different developmental stages reveal those genes to be expressed and differentially regulated in bladder development. Furthermore, those genes are differentially expressed in various bladder cancers with down-regulated genes in bladder development being up-regulated in bladder cancer and vice versa. These findings suggest genetic drivers for classic bladder exstrophy and their potential role in CBE-associated bladder cancer susceptibility.
The most significant CBE-associated locus is found on chromosome 5, housing the homeobox transcription factor ISL1 gene, known to be involved in the development of different tissues. Mouse models have been recently shown its role in urinary tract and genitalia development, and our RNA-seq also indicates high expression of ISL1 during early bladder developmental stages, followed by a strong decreased expression in differentiated bladder tissue. Exome sequencing of ISL1 in CBE cohort did not find any pathogenic variants in ISL1, suggesting that its contribution to CBE lies in gene regulation rather than genetic variation.
Our GWAS identified a regulatory variant rs2303751 in ISL1 that is suggested to be target of EZH2, recently shown to act as a transcription factor. Here, we describe a novel 1.2 kb intragenic promoter (called Fragment 2) residing between 6.2 and 7.4 kb downstream of the ISL1 transcription starting site. This promoter is active in the reverse DNA strand of ISL1 and in HEK 293 cells harbours a binding side for EZH2 where the rs2303751 marker reseeds. Here we show, that EZH2 enhance Fragment 2 activity and that its silencing reduces ISL1 expression. Further in vivo experiments in zebrafish ezh2-/- ko larvae display tissues specificity of ISL1 regulation with reduced expression that locates specifically in the pronephric region of zebrafish larvae. In addition, a shorter and malformed nephric duct is observed in ezh2-/- ko zebrafish Tg(wt1ß:eGFP) reporter lines.
Since in human Fragment 2 is active in the reverse orientation of ISL1, we showed that in HEK 293 its EZH2 regulation is not altering the expression of ISL1-DT, the immediate divergent transcript from ISL1, but rather disrupts the ratio of the ISL1/ISL1-DT cassette. Our study proposes that EZH2 is a key regulator of ISL1 during early urinary tract formation and suggests tissue specific ISL1 dysregulation as an underlying mechanism for CBE formation.
While the phenotype of CBE is well understood, the genetic and molecular causes remain largely unknown. Previous genetic studies have identified one locus associated with CBE, but these studies had limited sample sizes. The aim of this study is to identify new risk loci and gain a better understanding of their contributions to CBE and CBE-associated cancer. Furthermore, the study aims to elucidate the molecular mechanisms through which the associated ISL1 gene contributes to CBE.
For that, this study shows here the largest genome-wide association study (GWAS) conducted on CBE to date, identifying eight genome-wide significant loci, seven of which are novel. Within these loci, ten coding and four non-coding genes are found. RNA-seq analysis of mouse and human embryonic bladder tissues at different developmental stages reveal those genes to be expressed and differentially regulated in bladder development. Furthermore, those genes are differentially expressed in various bladder cancers with down-regulated genes in bladder development being up-regulated in bladder cancer and vice versa. These findings suggest genetic drivers for classic bladder exstrophy and their potential role in CBE-associated bladder cancer susceptibility.
The most significant CBE-associated locus is found on chromosome 5, housing the homeobox transcription factor ISL1 gene, known to be involved in the development of different tissues. Mouse models have been recently shown its role in urinary tract and genitalia development, and our RNA-seq also indicates high expression of ISL1 during early bladder developmental stages, followed by a strong decreased expression in differentiated bladder tissue. Exome sequencing of ISL1 in CBE cohort did not find any pathogenic variants in ISL1, suggesting that its contribution to CBE lies in gene regulation rather than genetic variation.
Our GWAS identified a regulatory variant rs2303751 in ISL1 that is suggested to be target of EZH2, recently shown to act as a transcription factor. Here, we describe a novel 1.2 kb intragenic promoter (called Fragment 2) residing between 6.2 and 7.4 kb downstream of the ISL1 transcription starting site. This promoter is active in the reverse DNA strand of ISL1 and in HEK 293 cells harbours a binding side for EZH2 where the rs2303751 marker reseeds. Here we show, that EZH2 enhance Fragment 2 activity and that its silencing reduces ISL1 expression. Further in vivo experiments in zebrafish ezh2-/- ko larvae display tissues specificity of ISL1 regulation with reduced expression that locates specifically in the pronephric region of zebrafish larvae. In addition, a shorter and malformed nephric duct is observed in ezh2-/- ko zebrafish Tg(wt1ß:eGFP) reporter lines.
Since in human Fragment 2 is active in the reverse orientation of ISL1, we showed that in HEK 293 its EZH2 regulation is not altering the expression of ISL1-DT, the immediate divergent transcript from ISL1, but rather disrupts the ratio of the ISL1/ISL1-DT cassette. Our study proposes that EZH2 is a key regulator of ISL1 during early urinary tract formation and suggests tissue specific ISL1 dysregulation as an underlying mechanism for CBE formation.
Klassifikation (DDC)
570 Biowissenschaften, BiologieZugehörige Publikation(en)
https://doi.org/10.1038/s42003-022-04092-3Mingardo, Enrico: Classic Bladder Exstrophy: identification of genetic markers and characterization of its associated ISL1 gene. - Bonn, 2024. - Dissertation, Rheinische Friedrich-Wilhelms-Universität Bonn.
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5-78561
Online-Ausgabe in bonndoc: https://nbn-resolving.org/urn:nbn:de:hbz:5-78561
@phdthesis{handle:20.500.11811/12273,
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5-78561,
author = {{Enrico Mingardo}},
title = {Classic Bladder Exstrophy: identification of genetic markers and characterization of its associated ISL1 gene},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2024,
month = sep,
note = {Classic bladder exstrophy (CBE) is a congenital anomaly with an occurrence of 1 in 30.000 new born. It is characterized by the protrusion of the bladder plate through an open abdominal wall, often accompanied by kidney and upper urinary tract abnormalities. Its long-term complications include different bladder malignancies.
While the phenotype of CBE is well understood, the genetic and molecular causes remain largely unknown. Previous genetic studies have identified one locus associated with CBE, but these studies had limited sample sizes. The aim of this study is to identify new risk loci and gain a better understanding of their contributions to CBE and CBE-associated cancer. Furthermore, the study aims to elucidate the molecular mechanisms through which the associated ISL1 gene contributes to CBE.
For that, this study shows here the largest genome-wide association study (GWAS) conducted on CBE to date, identifying eight genome-wide significant loci, seven of which are novel. Within these loci, ten coding and four non-coding genes are found. RNA-seq analysis of mouse and human embryonic bladder tissues at different developmental stages reveal those genes to be expressed and differentially regulated in bladder development. Furthermore, those genes are differentially expressed in various bladder cancers with down-regulated genes in bladder development being up-regulated in bladder cancer and vice versa. These findings suggest genetic drivers for classic bladder exstrophy and their potential role in CBE-associated bladder cancer susceptibility.
The most significant CBE-associated locus is found on chromosome 5, housing the homeobox transcription factor ISL1 gene, known to be involved in the development of different tissues. Mouse models have been recently shown its role in urinary tract and genitalia development, and our RNA-seq also indicates high expression of ISL1 during early bladder developmental stages, followed by a strong decreased expression in differentiated bladder tissue. Exome sequencing of ISL1 in CBE cohort did not find any pathogenic variants in ISL1, suggesting that its contribution to CBE lies in gene regulation rather than genetic variation.
Our GWAS identified a regulatory variant rs2303751 in ISL1 that is suggested to be target of EZH2, recently shown to act as a transcription factor. Here, we describe a novel 1.2 kb intragenic promoter (called Fragment 2) residing between 6.2 and 7.4 kb downstream of the ISL1 transcription starting site. This promoter is active in the reverse DNA strand of ISL1 and in HEK 293 cells harbours a binding side for EZH2 where the rs2303751 marker reseeds. Here we show, that EZH2 enhance Fragment 2 activity and that its silencing reduces ISL1 expression. Further in vivo experiments in zebrafish ezh2-/- ko larvae display tissues specificity of ISL1 regulation with reduced expression that locates specifically in the pronephric region of zebrafish larvae. In addition, a shorter and malformed nephric duct is observed in ezh2-/- ko zebrafish Tg(wt1ß:eGFP) reporter lines.
Since in human Fragment 2 is active in the reverse orientation of ISL1, we showed that in HEK 293 its EZH2 regulation is not altering the expression of ISL1-DT, the immediate divergent transcript from ISL1, but rather disrupts the ratio of the ISL1/ISL1-DT cassette. Our study proposes that EZH2 is a key regulator of ISL1 during early urinary tract formation and suggests tissue specific ISL1 dysregulation as an underlying mechanism for CBE formation.},
url = {https://hdl.handle.net/20.500.11811/12273}
}
urn: https://nbn-resolving.org/urn:nbn:de:hbz:5-78561,
author = {{Enrico Mingardo}},
title = {Classic Bladder Exstrophy: identification of genetic markers and characterization of its associated ISL1 gene},
school = {Rheinische Friedrich-Wilhelms-Universität Bonn},
year = 2024,
month = sep,
note = {Classic bladder exstrophy (CBE) is a congenital anomaly with an occurrence of 1 in 30.000 new born. It is characterized by the protrusion of the bladder plate through an open abdominal wall, often accompanied by kidney and upper urinary tract abnormalities. Its long-term complications include different bladder malignancies.
While the phenotype of CBE is well understood, the genetic and molecular causes remain largely unknown. Previous genetic studies have identified one locus associated with CBE, but these studies had limited sample sizes. The aim of this study is to identify new risk loci and gain a better understanding of their contributions to CBE and CBE-associated cancer. Furthermore, the study aims to elucidate the molecular mechanisms through which the associated ISL1 gene contributes to CBE.
For that, this study shows here the largest genome-wide association study (GWAS) conducted on CBE to date, identifying eight genome-wide significant loci, seven of which are novel. Within these loci, ten coding and four non-coding genes are found. RNA-seq analysis of mouse and human embryonic bladder tissues at different developmental stages reveal those genes to be expressed and differentially regulated in bladder development. Furthermore, those genes are differentially expressed in various bladder cancers with down-regulated genes in bladder development being up-regulated in bladder cancer and vice versa. These findings suggest genetic drivers for classic bladder exstrophy and their potential role in CBE-associated bladder cancer susceptibility.
The most significant CBE-associated locus is found on chromosome 5, housing the homeobox transcription factor ISL1 gene, known to be involved in the development of different tissues. Mouse models have been recently shown its role in urinary tract and genitalia development, and our RNA-seq also indicates high expression of ISL1 during early bladder developmental stages, followed by a strong decreased expression in differentiated bladder tissue. Exome sequencing of ISL1 in CBE cohort did not find any pathogenic variants in ISL1, suggesting that its contribution to CBE lies in gene regulation rather than genetic variation.
Our GWAS identified a regulatory variant rs2303751 in ISL1 that is suggested to be target of EZH2, recently shown to act as a transcription factor. Here, we describe a novel 1.2 kb intragenic promoter (called Fragment 2) residing between 6.2 and 7.4 kb downstream of the ISL1 transcription starting site. This promoter is active in the reverse DNA strand of ISL1 and in HEK 293 cells harbours a binding side for EZH2 where the rs2303751 marker reseeds. Here we show, that EZH2 enhance Fragment 2 activity and that its silencing reduces ISL1 expression. Further in vivo experiments in zebrafish ezh2-/- ko larvae display tissues specificity of ISL1 regulation with reduced expression that locates specifically in the pronephric region of zebrafish larvae. In addition, a shorter and malformed nephric duct is observed in ezh2-/- ko zebrafish Tg(wt1ß:eGFP) reporter lines.
Since in human Fragment 2 is active in the reverse orientation of ISL1, we showed that in HEK 293 its EZH2 regulation is not altering the expression of ISL1-DT, the immediate divergent transcript from ISL1, but rather disrupts the ratio of the ISL1/ISL1-DT cassette. Our study proposes that EZH2 is a key regulator of ISL1 during early urinary tract formation and suggests tissue specific ISL1 dysregulation as an underlying mechanism for CBE formation.},
url = {https://hdl.handle.net/20.500.11811/12273}
}
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