The Development Of 22q11.2 Copy Number Variation Detection Method And The Investigation Of GATA4 Gene Mutation In Patients With Congenital Heart Disease

Abjective:The genomic copy number variations(CNVs)and mutations on the genes related to heart development are two of the most crucial machasims involved in congenital heart disease(CHD).Copy number variation is a common cause of cardiac malformation and a large proportion of patients with CHD have 22q11.2 microdeletions.GATA4 is an early cardiac cell marker during the embryonic development.As a highly conserved transcription factor,GATA4 regulates a variety of physiological processes,especially the cardiac development.This study was to develop two novel methods for22q11.2 CNV detection and clarify the potential association of the GATA4 mutation with congenital heart disease in a CHD family.Methods:1.We developed a novel technique,CNVplex~?,for high-resolution detection of22q11.2 CNV related diseases.The performance of this method was validated by known cases.Furthermore,CNVplex~?was used to screen for the sub-chromosomal imbalances in 818 CHD patients to determine the frequency of 22q11.2 deletion among sub-groups of CHD.2.By using the multiplex PCR with different primer pairs and limiting deoxynucleotide triphosphates(d NTPs),we developed a simple method for the22q11.2 microdeletion detection.This method was validated by 100 known cases which were extensively characterized to test if the new system could be applified in the large population screening.3.Based on the gene structure and functional characteristic of GATA4,we evaluated the pathogenicity of p.R311W mutation by testing its intracellular distribution,the ability of DNA binding and the transcriptional activity to the downstream genes.Results:1.In the methodology development phase,CNVplex~? confirmed all the copy number aberrations that were previously identified by other methods,demonstrating 100%sensitivity and specificity.In the validation phase,22q11.2 deletion was detected in 39out of 818 CHD patients(4.8%).Our data demonstrated that the frequency of 22q11.2deletion varied among sub-groups of CHD patients.Noticeably,the 22q11.2 deletion was more commonly observed in cases with conotruncal defects(CTD)than in cases with non-CTD.2.By using the HRM technology,we could assess the copy number variations of CLTCL1、KLHL22、PI4KA、SNAP29 genes which were located in different low copy number repeat regions.This new method could be used for 22q11.2 microdeletion detection with 100%sensitivity and specificity.3.The cross-species alignment of multiple GATA4 protein sequences showed that the altered amino acid(p.R311)was completely conserved evolutionarily.Meanwhile,functional analysis displayed that the p.R311W mutation didn’t change its normal nuclear distribution but was associated with a decreased transcriptional activity.The p.R311W mutation had altered the charge of the residue and could be the genetic susceptible basis in this study.Conclusion:CNVplex~? and HRM were sensitive and specific in the detection of CNVs.Considering its high resolution,CNVplex~?could be used in the detection of copy number variations in 22q11.2 and other diseases with similar phynotype.Restricting the d NTP concentration with HRM is a simple way to assess the CNVs and can be used in the large population screening.A novel GATA4 mutation,c.C931T(p.R311W),was identified and functionally analyzed in our research.The in vitro assays of the mutation resulted in a diminished transactivation of downstream target genes and suggested that the functionally impaired GATA4 contributed to CHD in this family.