< Back to previous page
Project
Prenatal Chromosomal Microarray Analysis and Identification of Genetic Variants in Congenital Diaphragmatic Hernia.
Chromosomal microarray analysis has gradually replaced conventional karyotyping over recent years in the postnatal setting which has revolutionized whole genomescreening for genomic imbalances in patients. We sought to evaluate the benefits and the challenges of applying chromosomal microarrays to prenatal diagnosis for referrals with abnormal ultrasound findings. Our findings, presented in Chapter 3, demonstrate a diagnostic yieldof ~10%. Importantly, ~3% are caused by submicroscopic CNVs whichwould go undetected by conventional karyotyping alone. Furthermore, the higher resolution offered by chromosomal microarray analysis led to important additional information in ~4% of patients. Of particular interest we discover a novel and unexpected advantage of arrays; a 500kb paternal insertional translocation is the likely driver of a de novo unbalanced translocation, thus improving recurrence risk calculation in this family. Our study has, in part, paved the way for the recent Summary Guidelines for Prenatal Chromosomal Microarray Analysis and Genetic Counselling from the Belgian Society for Human Genetics [http://www.beshg.be/download/annex_2_summary_of_array_and_prenatal_guidelines_20130502.pdf]. The implementation of prenatal chromosomal microarray analysis as the first tier test in place of conventional karyotyping brings the standard of prenatal diagnosis in line with that which is provided for postnatal genetic diagnosis.
Congenital diaphragmatic hernia(CDH) is a life-threatening prenatal disorder detectable by ultrasound during pregnancy. We sought to further unravel the genetic factorsunderlying isolated CDH by the design of a custom microarray covering genomic loci recurrently associated with CDH and candidate CDH genes. Our retrospective screen of 79 isolated CDH patients using this custom microarray is presented in Chapter 4. This study identified a novel duplication of the EFNB1 gene in a male patient which was consideredlikely to be pathogenic. Since our publication, a second case of a male CDH patient with duplication of EFNB1 was reported, thus reinforcing EFNB1 dosage sensitivity as a cause of isolated CDH. In order to further identify (novel) CNVs and genes associated with isolated CDH,we undertook a prospective prenatal study using chromosomal microarrayswith genome-wide coverage in 75 foetuses with isolated CDH, which is presented in Chapter 4. This study revealed submicroscopic de novo pathogenic CNVs in 9.3% and rare inherited variants which may be involvedin CDH in a further 4% of foetuses. This diagnostic yield is significantly higher than the ~3% rate of pathogenic submicroscopic CNVs which we observed in our prenatal study using the same microarray platform. Isolated CDH thus represents a valid cohort for CNV screening in the prenatal phase, and suggests that the clinical utility of conventional karyotyping is questionable for this group of patients. This study allowed us to further refine the critical region at 15q26 to only 2 genes, pinpointing NR2F2 as the causal gene. We add further evidence for the 15q25.2 and 16p11.2 recurrent microdeletions as CDH loci, andwe identify novel CNVs not previously observed in association with CDH,including a duplication of 4p15.2-p14.
We next evaluated the use of exome sequencing for the investigation of isolated CDH and non-isolated CDH where a genetic cause was suspected. Our results show that exome sequencing represents an effective technique with which to investigate familial CDH, described in Chapter 5. In the first family studied, we identified a nonsense mutation in ZFPM2 in 2 individuals with isolated CDH, as well as a sibling with a congenital heart defect. Surprisingly, the mutation was shown to be transmitted from the unaffected mother, and is also carried by the maternal grandfather and the maternalsister, both of whom are also asymptomatic. This intriguing finding highlights the complexity of CDH, reinforcing the involvement of additional as yet unidentified (epi)genetic factors in CDH penetrance. In a second family with 2 male foetuses with MCA, we identify a mutation in the X-linked PORCN gene inherited from an unaffected mother who wasshown to have extreme skewing of X-inactivation. This further implicates Wnt signaling as playing a role in CDH, as well as multiple aspects of foetal development. In a third consanguineous family we identify a mutation in PIGN in a foetus with MCA, inherited from carrier parents. PIGN is involved in GPI anchor synthesis and our finding adds to a growing body of evidence that defective GPI anchor synthesis causes multiple phenotypes in humans.
Given the variation in severity ofherniation and thus pulmonary hypoplasia, as well as differences in responses to foetal and / or neonatal therapy for CDH patients, we sought to explore whether gene expression analysis of amniotic fluid cells from CDH foetuses could identify dysregulated genes and biological pathways which may act as predictive biomarkers. In this exploratory study we applied RNA-sequencing to investigate gene expression in cultured cells sourced from amniotic fluid of isolated CDH patients, described in Chapter 6. This analysis identifies 2 potential molecular subtypes ofisolated CDH, one of which is characterized by downregulation of TGFB1 and CTGF, and upregulation of TNF, IL6 and IL8. This highlights downregulation of TGFB signalling as a likely cause of much of of the downstream dysregulation in gene expression observed, including that of CTGFwhich has been previously implicated in the nitrofen rodent model of CDH. Furthermore, this group of patients shows an apparent inflammatory response indicated by the upregulation of TNF, IL6 and IL8 which mayin turn exacerbate postnatal pulmonary hypertension. These findings direct future targeted studies in a larger cohort of isolated CDH patients to determine the clinical significance and therapeutic potential.
</></></>
Congenital diaphragmatic hernia(CDH) is a life-threatening prenatal disorder detectable by ultrasound during pregnancy. We sought to further unravel the genetic factorsunderlying isolated CDH by the design of a custom microarray covering genomic loci recurrently associated with CDH and candidate CDH genes. Our retrospective screen of 79 isolated CDH patients using this custom microarray is presented in Chapter 4. This study identified a novel duplication of the EFNB1 gene in a male patient which was consideredlikely to be pathogenic. Since our publication, a second case of a male CDH patient with duplication of EFNB1 was reported, thus reinforcing EFNB1 dosage sensitivity as a cause of isolated CDH. In order to further identify (novel) CNVs and genes associated with isolated CDH,we undertook a prospective prenatal study using chromosomal microarrayswith genome-wide coverage in 75 foetuses with isolated CDH, which is presented in Chapter 4. This study revealed submicroscopic de novo pathogenic CNVs in 9.3% and rare inherited variants which may be involvedin CDH in a further 4% of foetuses. This diagnostic yield is significantly higher than the ~3% rate of pathogenic submicroscopic CNVs which we observed in our prenatal study using the same microarray platform. Isolated CDH thus represents a valid cohort for CNV screening in the prenatal phase, and suggests that the clinical utility of conventional karyotyping is questionable for this group of patients. This study allowed us to further refine the critical region at 15q26 to only 2 genes, pinpointing NR2F2 as the causal gene. We add further evidence for the 15q25.2 and 16p11.2 recurrent microdeletions as CDH loci, andwe identify novel CNVs not previously observed in association with CDH,including a duplication of 4p15.2-p14.
We next evaluated the use of exome sequencing for the investigation of isolated CDH and non-isolated CDH where a genetic cause was suspected. Our results show that exome sequencing represents an effective technique with which to investigate familial CDH, described in Chapter 5. In the first family studied, we identified a nonsense mutation in ZFPM2 in 2 individuals with isolated CDH, as well as a sibling with a congenital heart defect. Surprisingly, the mutation was shown to be transmitted from the unaffected mother, and is also carried by the maternal grandfather and the maternalsister, both of whom are also asymptomatic. This intriguing finding highlights the complexity of CDH, reinforcing the involvement of additional as yet unidentified (epi)genetic factors in CDH penetrance. In a second family with 2 male foetuses with MCA, we identify a mutation in the X-linked PORCN gene inherited from an unaffected mother who wasshown to have extreme skewing of X-inactivation. This further implicates Wnt signaling as playing a role in CDH, as well as multiple aspects of foetal development. In a third consanguineous family we identify a mutation in PIGN in a foetus with MCA, inherited from carrier parents. PIGN is involved in GPI anchor synthesis and our finding adds to a growing body of evidence that defective GPI anchor synthesis causes multiple phenotypes in humans.
Given the variation in severity ofherniation and thus pulmonary hypoplasia, as well as differences in responses to foetal and / or neonatal therapy for CDH patients, we sought to explore whether gene expression analysis of amniotic fluid cells from CDH foetuses could identify dysregulated genes and biological pathways which may act as predictive biomarkers. In this exploratory study we applied RNA-sequencing to investigate gene expression in cultured cells sourced from amniotic fluid of isolated CDH patients, described in Chapter 6. This analysis identifies 2 potential molecular subtypes ofisolated CDH, one of which is characterized by downregulation of TGFB1 and CTGF, and upregulation of TNF, IL6 and IL8. This highlights downregulation of TGFB signalling as a likely cause of much of of the downstream dysregulation in gene expression observed, including that of CTGFwhich has been previously implicated in the nitrofen rodent model of CDH. Furthermore, this group of patients shows an apparent inflammatory response indicated by the upregulation of TNF, IL6 and IL8 which mayin turn exacerbate postnatal pulmonary hypertension. These findings direct future targeted studies in a larger cohort of isolated CDH patients to determine the clinical significance and therapeutic potential.
</></></>
Date:1 Jun 2009 → 14 Apr 2014
Keywords:prenatal diagnosis, congenital diaphragmatic hernia, molecular karyotyping, array CGH
Disciplines:Genetics, Systems biology, Molecular and cell biology
Project type:PhD project