Introduction:
Congenital heart disease (CHD) is one of the most common birth defects and a prime reason for deaths in first year of life. It accounts for about 20% of congenital defects in infants. The understanding of this study is based on evolution of heart, heart development in mammals and molecular regulation of cardiac development.
Evolution of heart saw change in the number of chambers, with fishes (two chambers), amphibians (three chambers) and reptiles, birds and mammals (four chambers). During the developmental process, heart is generated from splanchnic mesoderm that contains the original cardiac cells.
Mouse has a gestation period of 20-21 days but similar stages of human heart development. At 7.5 days of development, the cardiac crescent and cardiac progenitor cells migrate to the sides where gastrulation starts. At 8.5 days, the layer fuses with the mid-line forming a heart tube (with an atrium and a ventricle). At 9.5 days, the location of chambers is defined. Finally, at 14.5 days the heart formation is complete. Contributors of the heart are heart fields and cardiac neural crest cells (cNCC). Heart field is a group of cells that can receive the signals from around the tissue and are pluripotent. The cNCCs migrate to different locations to form different regions in the heart.
During molecular regulation, different markers in progenitor cells receive signals (regulating positively or negatively) from surrounding cells to go through various signalling pathways. For determination, gene NK2.5 marks which cell has the potential to become heart cells along with many other factors. Proliferation is controlled by growth and regulation factors. Differentiation is also regulated by a set of factors, generated by respective genes. The process of genetic regulation is not governed by one factor and it is an inter-regulated event in which many factors are working together with the steps overlapping.
Objective:
This study tries to understand the network of genes that control the development of heart, thus predicting the cellular and molecular cause of Congenital Heart Diseases.
Procedure (Method):
For this study, mouse models made by genetically modified mouse line (using genetic tools) were used. Human phenotype information was acquired from DNA samples of patients with symptoms. Combining these two informations, we get the gene expressions which form the molecular basis of this defect. Screening, validation and mechanism of regulation give the candidate gene for Human CHD.
The syndrome (22q11 syndrome or DiGeorge syndrome) under study is a deletion on 22nd chromosome, where the gene has only one copy of it. It also has facial defect symptoms, mental disorders and ear problems apart from the cardiac defect.
The mouse heart at 14.5 days is taken and evaluated for defect and analysed as what stage of development was compromised and which gene is responsible behind the defect. Our of the three gene alleles, different combinations can be used to decide certain dosage of the gene in the embryo.
DNA sample from the patient (with the deletion) was taken and exon sequencing (following qPCR) was performed to get the candidate gene. Genetic crossing was done for alleles to get allelic series, a combination of alleles in all possible permutations, and see the defects by isolation of embryonic heart at 15.5 days. Cardiac phenotype and gene expression study was performed to study the effects of different genes involved.
Results:
In this study, Tetralogy of Fallot (aortic valve and pulmonary vale being at same level) was found to be more frequent in Crkl knock-outs. An opening between right and left ventricles was also observed. OFT defects (two arteries, aorta and pulmonary artery, connected to the right ventricle) were found more often in NEO/- than other variants. It was less severe in NEO/NEO.
AV cushion defect was found to be dosage sensitive hypertrophic cushions were found in knock-outs compared to wild type along with primary defects in the aorta. High frequency of muscle defects were also observed in knock-outs compared to control.
Gene expression study was performed by pooling the heart tube tissues for different samples and performing qPCR for CrkL related genes (27 genes were selected in this study). The expression study showed erbb3 and ror2 were down-regulated which is very much involved in the heart defects.
Conclusion:
The following conclusions can be drawn from the study.
- Human hemizygous for CRKL have OFT and septation defects
- The models supplements for the scarcity of true knockouts in humans
- Crkl dosage has an effect on variation of heart phenotype and severity
- Crkl/NEO/- allelic combination is very useful in studying DORV phenotype.
- Crkl mutants showed more varied phenotypes and were involved in more than one cardiac lineage, secondary heart field and endocardial lineage.
- Erbb3 gene effects both human and mouse.
