Gene Mutation In Congenital Diaphragmatic Hernias (CDH)

A Congenital Diaphragmatic Hernia is a birth defect that causes a tear in the diaphragm that pushes abdominal organs into the chest cavity, therefore, preventing proper lung development. CDH occurs in one out of 3,000 live births, and has a high mortality and morbidity rate (POBER 2007), with survivors requiring high technology-driven interventional perinatal care (STOLAR 2012) in tertiary or quaternary neonatal intensive care units. 40% of CDH patients have multiple anomalies in addition to CDH, but as many as 60% are isolated with the potential for increased survival if the associated lung hypoplasia responsible for its high morbidity can be improved. If substantial pulmonary differentiation can be induced to occur in the lung postnatally, the use of ExtraCorporeal Membrane Oxygenation (ECMO)(WILSON et al. 1994; STOLAR 2012; GARCIA 2014; BEAUMIER et al. 2015) and aggressive ventilatory management (SNOEK et al. 2015) may be reduced, thus protecting fragile lungs from secondary barotrauma and bronchopulmonary dysplasia. Due to improved medical and surgical care, CDH survival, particularly for that subset of patients who are diagnosed in utero and receive care in specialized Neonatal Intensive Care Units at tertiary and quaternary centers, can now reach as high as 80%(POBER 2007; POBER 2008; POBER et al. 2010). Even so, among all fetuses and infants with CDH, over half still die from severe pulmonary hypoplasia and pulmonary hypertension, while long-term survivors experience varying degrees of pulmonary insufficiency and multi-organ morbidity (SNOEK et al. 2016).

The overall research strategy for Gene Mutation and Rescue in Human Diaphragmatic Hernia is to identify gene variants in patients with Congenital Diaphragmatic Hernia (LONGONI et al. 2014) from genomic analyses, test high priority candidates for validation and function in vitro and ex vivo; in animal models, determine molecular pathways defined by integrating already known and new candidate genes, and lastly, select molecular targets from which to conceive postnatal treatment strategies for CDH.

Based upon our preliminary studies, there has been convincing evidence that CDH is genetically heterogeneous; however, if studies were to continue the overall objective would be to show that the genes responsible will fall into shared or parallel molecular pathways that will impute therapeutics that might act effectively downstream of these causative pathways. This strategy would focus on prioritizing variants by the involvement of their wild type counterparts in normal development of lung and diaphragm with the hypothesis that genes causing CDH will be important for normal development.

According to research conducted by Longoni 2014, a large sample size CDH cohort was recruited to accumulate: clinical data, biospecimens, and sequencing data for probands and parents (trios), and multiplex families, which were further interrogated by bioinformaticians who are equipped to analyze collected data via specialized algorithms. After extensive sequencing and analytical prioritization, multiple candidate variants require validation, functional analyses, and replication in other human cohorts. Candidate sequence variants generated from analyses of single nucleotide variants (SNV) and copy number variations (CNVs) in CDH probands and trios were combined with genes already known to cause CDH in mice and humans and with those uncovered by unbiased reverse genetic screens resulting from the knockout mouse experiments (Longoni 2014). From this validated group of genes, those that produce a diaphragm and lung defect, as detected by microCT, then undergo functional studies in a variety of in vitro and ex vivo assays. At all stages, high priority candidates are integrated by bioinformatic placement into protein interaction networks to help uncover interacting, perturbated molecular pathways. Highest priority (ranked) candidate variants were knocked out by gene editing and the defective lungs of the animal models generated were studied for rescue in vitro and in vivo by genes, proteins, or their mimetics, as directed by functional analyses.

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Pober 2007 suggests that there is a great need for functional validation of the candidate genes’ influences on lung and diaphragm development in mouse models. As a result, there is a collaborative effort across many studies to determine if, where, and how a particular variant fits into the complex heterogeneity now known to be characteristic of a birth defect such as CDH. To help remedy this gap in knowledge, many found research studies have turned to satellite CDH sites that have the skills and the experience to examine embryonic development by histology, immunohistochemistry (IHC), in situ hybridization (ISH), morphometrics, and specific assessment of alveolar and vascular morphology and function, such as in the Fgfr3:4 Cre-directed lung hypoplasia model, and the Pbx1:2 Cre-directed pulmonary hypertension model. Moreover, data analyzed from sed institutions have helped establish a number of in vitro and ex vivo validation assays to test the function of new candidate variants, which can also serve as secondary screens for molecules emanating from sequence variants and from small molecule screens designed and executed.

The phenotypes of newborns with CDH are heterogeneous with significant variability in severity, response to treatment, and long-term outcomes (POBER 2007). Prior evidence, particularly from nitrofen treatment and vitamin A deficiency models, has suggested that environmental and metabolic factors may also play important roles in the cause of CDH. This may be due to epigenetic factors, which can be investigated in future studies in human patients and mouse models, that have identified key regulators and mechanisms of diaphragm and lung development- abnormalities of which can lead to pulmonary hypertension and failure of alveolar differentiation that cause death or long term morbidity in many patients with CDH. While some of the genetic mutations that result in CDH impair only formation of the diaphragm, several of the genes and pathways implicated in diaphragm development are also required for normal development of epithelial, vascular, and interstitial components of the lung, and it is those that affect both lung and diaphragm that cause the most morbidity.

Therefore, research studies have sought to define a number of genetic causes of CDH by use of genetic and genomic platforms, such as high density array Comparative Genomic Hybridization (aCGH) to detect copy number variations (CNVs) and WES and WGS to detect single nucleotide variants (SNVs), both inherited and de novo (STOLAR 2012). The overarching goal seeks to converge the multiple genes detected by studies in patients and model organisms into cogent overlapping molecular pathways in order to identify potential therapeutic targets. Moreover, the first sub-goal would be to focus on treatments that can be administered postnatally, where septation and vascular remodeling may yield realistic therapeutic targets (SNOEK et al 2016). However, it is important to note that gene variants, such as those leading to early branching defects which are embryonically lethal if homozygous, may be sublethal if allelic, or if combined with protective variants as modifiers, making these variants also amenable as therapeutic targets.

According to Longoni 2014, there exists biospecimen and data repositories where over 500 clinical data points have been collected on each known CDH subject including: prenatal and family history, additional birth defects, surgical history, feeding, respiratory, medication and discharge status. Moreover, blood, saliva, or skin biopsy during surgery for CDH repair is collected from all participants, the biological parents, and any other affected relative. Likewise, diaphragm biopsies are also collected at the time of diaphragm repair, and echocardiograms at 1 and 3 months are read for pulmonary hypertension by a single cardiologist (Longoni 2014). In addition, multigenerational family histories also seem to be collected and patients are followed longitudinally with formal developmental assessments at 2 and 5 years of age.

Future studies point towards discovering treatments appropriate for the immediate postnatal period to improve clinical outcomes by either avoiding or shortening the use of ECMO and high frequency ventilation, which carry their own set of severe therapy-associated complications, in addition to the pulmonary hypoplasia and pulmonary hypertension extant in the disease itself. Moreover, there is a need to employ genomic methods such as Whole Exome Sequencing (WES), array Comparative Genomic Hybridization (aCGH), and Whole Genomic Sequencing (WGS), and to integrate variant findings with data from expression analyses of developing diaphragm and lung tissues in mouse models, and with diaphragm biopsies from human CDH patients. It is the goal to use a growing cohort and an analytic approach to generate candidate genes that would yield important clues as to the molecular pathogenesis of CDH, particularly after integrating the highest priority candidates into molecular pathways.