| Research Background:Congenital heart disease(CHD)is one of the most common and most serious congenital malformations.According to the”China Birth Defect Prevention Report",the incidence of perinatal CHD based on the National Birth Defect Monitoring System in 2011 was as high as 40.95/10000,ranking top list in birth defects of China.If these newborns with CHD do not undergo intervention and treatment timely,about one-third of them will die in the first month after birth due to serious illness and deformity.According to statistics,the social and economic burden caused by emerging CHD is more than 12.6 billion yuan per year.CHD brings great adverse impact to society and families.Epidemiological studies have shown that the combination of environmental factors and genetic factors leads to the occurrence of CHD,in which genetic factors play an important role.Exploring the genetic factors of CHD can help us to know about CHD deeply.Genetic analysis of CHD can not only clarify the genetic background of CHD but also provide important information for genetic counseling,risk assessment of reproduction,Prognosis of CHD fetus and the judgment of surgical outcome.Objective:This study was conducted on fetuses with congenital cardiovascular malformations,including cases of live births,stillbirths,births,and unborn fetuses.Our research can overcome shortages such as rare type of CHD by selectiong live birth’s cases with CHD and let us understand comprehensively the pathogenesis of CHD,and expect to find new genetic variations which can cause CHD.In addition,we try to investigate the relationship between the phenotype and genotype of CHD.Method:(1)Collecting amniotic fluid,cord blood,umbilical cord or fetal tissues of fetuses who present cardiac malformations in prenatal ultrasound;(2)According to whether combined with extracardiac abnormalities,cases of CHD were divided into three groups:isolated CHD group(group A),CHD combined with extracardiac malformation group(group B),and CHD combined with abnormality of ultrasound soft index group(group C).(3)Both G-banding karyotype analysis and chromosome microarray analysis(CMA)were performed on amniotic fluid and cord blood samples,and CMA was performed on umbilical cord and fetal tissues samples.And whole exome sequencing was performed for some of those fetuses who detected no pathogenic or likely pathogenic variation by G-banding karyotype and CMA testing.Result:(1)117 f’etuses were detected with chromosomal aberrations(29.8%)in cases of 393 CHD,oi which 83 cases(21.1%)have pathogenic or likely pathogenic chromosomal aberrations.34 cases(8.7%)have chromosomal aberrations of unknown clinical significance.Among 83 fetuses who have pathogenic or likely pathogenic chromosomal aberrations,38 fetuses have numerical abnormalities of chromosome,12 fetuses have imbalanced chromosome translocations,12 fetuses were diagnosed as 22q11.2 microdeletion syndrome,5 fetuses were diagnosed as Jacobsen syndrome(-11q23 microdeletion).,1 fetus were diagnosed as wiliiams-Beuren syndrome(7q11.23 microdeletion).The remaining cases with pathogenic or likely pathogenic CNVs included 12 microdeletions,2 microduplications,and 1 complex variation in chromosome 2.(2)Karyotype analysis was performed on 352 specimens(including 41 cases of aborted fetuses).The detection rate of pathogenic or likely pathogenic variations is 16.5%(58/352).There are 2 cases witfh unknown-significance variations.the detection rate of chromosomal aberrations by karyotype analysis is 17.0%(60/352);CMA analysis was performed on 390 fetuses(3 cases failed to be perform CMA analysis for poor DNA quality),The detection rate of pathogenic or likely pathogenic variations was 20.5%(80/390)and 32 fetuses exist unknown-significance variations.The detection rate of CMA for all chromosomal aberrations is 28.7%(112/390).The detection rate of pathogenic and likely pathogenic variations of CMA is higher than that of karyotype(16’5%vs 20.5%),but the difference has no statistical significance(P>0.05).The detection rate of all abnormal variations including unknown significance of CMA was higher than that of karyotype(17.0%vs 28.7%)9 and the difference was significant statistically(P<0.05).(3)Among the 393 CHD fetuses,the fetuses of group A,B and C account for 64.1%(252/393),29.5%(116/393)and 6.4%(25/393)respectively.The detection rates of pathogenic and likely pathogenic variations were 13.9%(35/252),36.2%(42/116),and 24.0%(6/25)respectively.The detection rates of unknown-significance variations were 9.9%(25/252)and 6.0%(7/116)and 8.0(2/25)respectively.The detection rates of all chromosomal aberrations were 23.8%,42.2%and 32.0%respectively.Whetiher the detection rate of pathogenic and likely pathogenic variations,or the detection rate of all chromosomal aberrations,group B>group C>group A,and the difference between group A and B is significant statistically(P<0.05),but there is no statistically significant difference between group A and C,group B and C(P>0.05).(4)Whole exome sequencing was performed for 28 fetuses with congenital heart disease but no chromosomal aberration was detected by CMA and karyotype.Among which,13 likely pathogenic or pathogenic gene mutations are detected,3 unknown-significance gene mutations are detected,12 fetuses exist no gene mutation correlated with congenital heart disease.Conclusion(1)In CHD fetuses,the incidence of chromosomal aberrations(including aneuploidy,structural abnormalities,and genome copy number variations)is 29.8%.And whether isolated CHD or CHD with extracardiac abnormalities,chromosomal aberrations can be detected.Indicating that chromosomal variations are correlated with congenital heart malformation closely.(2)Compared with isolated CHD,the incidence of chromosomal aberrations is higher in CHD accompanied with extracardiac structural abnormalities or accompanied with abnormal ultrasound soft index.(3)Comparison of two techniques:CMA has higher resolution,locate the position of breakpoint more accurately,and can detect uniparental disomy.So CMA can significantly improve the detection rate of chromosomal aberrations.While karyotype analysis is the gold standard technique of mosaicism in low level,besides,it can help to determine the source and the type of the variations.Both techniques have their own advantages and disadvantages,so the combination of the two techniques will contribute to detect and analyze the chromosomal variations in CHD fetus more comprehensively.(4)Apart from known pathogenic and likely pathogenic chromosomal aberrations,chromosomal aberrations of unknown significance account for 8.7%CHD fetuses,suggesting those variations of unknown significance may play a role in congenital heart defects.(5)The etiology of CHD is very complicated.In addition to chromosomal aberrations,monogenic mutations can also be the causes of CHD.Accordingly,whole exome sequencing should be performed if no pathogenic or likely pathogenic chromosomal aberrations were detected by karyotype and CMA analysis,and further explore the genetic mechanism of the pathogenesis of CHD. |
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