Verification Of ZNF41and SPARC As Serum Candidate Biomarkers For Ventricular Septal Defect

Congenital heart disease (CHD) is one of the most common diseasesin birth defects. Ventricular septal defect (VSD), as the main type of congenitalheart disease, plays an important impact on the infants’ survival and quality oflife. In order to get a comprehensive understanding of the VSD’s complicatedetiology and mechanism, it not only needs to study the traditionally relatedsusceptibility genes and environmental factors, but also needs to study theproteins and signaling pathways controlled the heart abnormal differentiation,thus providing clues to disease development and disease diagnosis.This study was based on our previous research result that was a seriesof potential serum protein biomarkers of VSD by utilizing isobaric tags forrelative and absolute quantitation(iTRAQ) labeling combined with liquidchromatography and matrix-assisted laser desorption ionization tandem time offlight mass spectrometry (LC-MALDI-TOF/TOF MS) technology, and coupledwith the analysis of literature retrieval. The study utilized Western blot andenzyme linked immunosorbent assay (ELISA) to detect zinc finger protein41(ZNF41), secreted protein acidic and rich in cysteine (SPARC) expression levels in the serum samples, and used bioinformatics technology to analyze theirprotein structures and functions. This research was divided into two parts tovalidate whether ZNF41, SPARC could be used respectively as a serumcandidate biomarker of VSD. The purpose of this study was to provide thereference for early VSD diagnosis and pathogenesis research.PART1VERIFICATION OF ZNF41AS A SERUMCANDIDATE BIOMARKER FOR VENTRICULARSEPTAL DEFECTObjective To validate whether ZNF41can be used as a serum candidatebiomarker of VSD, and to analyze its protein structure and functions bybioinformatics.Methods The serum samples were collected from20objects with VSD, ASD(atrial septal defect), TOF (tetralogy of Fallot) and healthy controls, respectively.All the subjects were less than10years old. The study utilized Western blot andELISA to detect ZNF41expression levels in the serum samples, and usedbioinformatics technology to analyze its structure and functions, and theinvolved signaling pathways, etc.Results1. There was no statistically difference in the distributions of age,gender, and nationality among the VSD group, the ASD group, the TOF groupand healthy control group, respectively (P>0.05).2. Western blot results showed that ZNF41was detected in all samplesof research objects. However, the relative expression level of ZNF41in VSD group was higher than that in the other three groups (P<0.05).3. The results of ELISA indicated that the serum concentrations ofZNF41in VSD, ASD, TOF and healthy control group were (136.72±56.44)pg/ml,(94.54±41.98) pg/ml,(100.69±37.08) pg/ml, and (82.08±42.46) pg/ml,respectively. There were statistically significant differences among the fourgroups (P<0.05). Given the further comparisons between each two groups, therewere statistically significant differences between VSD group and the other threegroups respectively (P<0.05). Besides, there was no statistically significantdifference between each two groups in the other three groups (P>0.05).4. Bioinformatics analysis showed that ZNF41was the member ofC2H2zinc finger protein family. ZNF41could regulate the DNA-bindingactivities, could regulate gene expressions and protein generations, affecting celldifferentiations and organ developments, etc.Conclusion The concentration of ZNF41is higher in the serum of the VSDpatients, which suggests that ZNF41may be used as a serum candidatebiomarker for VSD. But the subsequent large sample clinical qualification andthe function researches of ZNF41are still needed. PART2VERIFICATION OF SPARC AS A SERUMCANDIDATE BIOMARKER FOR VENTRICULARSEPTAL DEFECTObjective To validate whether SPARC can be used as a serum candidatebiomarker of VSD, and to analyze SPARC by bioinformatics technology.Methods The serum samples were obtained from20objects with VSD, ASD,TOF and healthy controls, respectively. All the objects were less than10yearsold. The study used Western blot and ELISA to detect SPARC expression levelsin the serum samples, and utilized bioinformatics technology to predict itsstructure and functions.Results1. There was no statistically difference in the age, gender, andnationality among the four groups, respectively (P>0.05).2. Western blot analysis showed that SPARC was detected in allsamples. However, the relative expression level of SPARC in VSD group washigher than that in the other three groups (P<0.05).3. The ELISA results indicated that the serum concentrations ofSPARC in VSD, ASD, TOF and healthy group were (198.03±87.26) pg/ml,(137.02±83.89) pg/ml,(140.61±79.98) pg/ml and (91.30±55.50) pg/ml,respectively. There were statistically significant differences among the fourgroups (P<0.05). Given the further comparisons between each two groups, therewere statistically significant differences between the VSD group and the otherthree groups respectively (P<0.05). Besides, there was no statistically significantdifference between each two groups in the other three groups (P>0.05).4. Bioinformatics results showed that SPARC, with its uniquestructure domains, could affect cell differentiations and signal transductions, and participate in the development of organs such as heart, etc.Conclusion The concentration of SPARC is higher in the serum of the VSDpatients, which suggests that SPARC may be used as a serum candidatebiomarker for VSD. But the third large sample clinical qualification and SPARCbiological characteristics researches are still needed.