Analysis Of Chromosomal Aberrations In Fetuses With Congenital Heart Malformations

Research Background:Congenital heart malformations,also called congenital heart disease(CHD), are the most common birth defects. They account for one third of the birth defects and occurs in almost 6-8‰ live births. It is the leading cause of neonatal and childhood death. CHD is also the major cause of miscarriages and at least 10% fetuses aborted within the first trimester have congenital heart malformations, so it is regarded as an important reason leading to early abortion. So far, over 75% patients who have CHD can survive to adulthood depending on the advance of prenatal diagnosis,cardiac surgical techniques and cardiac care. It is estimated that the incidence of CHD in adults is 3‰ with growing trend. By investigating the evolution and morphologic of embryo heart, it can help us to understand the pathogenesis of CHD, to improve prenatal diagnosis and health management, to provide better treatment of CHD and to prevent the severe birth defects.The etiology of CHD is complex. So far, only 20% of CHD patients can be found to have clear pathogenesis and the rest of 80% CHD patients have unknown reason. The main causes leading to CHD include environmental and genetic factors as well as the interaction between environmental and genetic factors. The environmental factors associated with CHD include fetal infection such as TORCH pathogenic microorganisms, teratogens exposures(retinoic acid, lithium, phenytoin sodium, alcohol), maternal diseases(diabetes, systemic lupus erythematosus). The genetic factors such as chromosomal numerical or structural anomalies, Mendelian syndrome and non-syndromal single gene mutations are the most common reasons of CHD.50% patients with Down syndrome and Turner syndrome, more than 90% patinets with Edwards syndrome and Patau syndrome have congential heart malformations. Over forty different kinds of chromosomal microdeletion or microduplication syndromes were reported to be associated with CHD. More than 200 genes are linked to CHD, which include syndromic and non-syndromic CHD. Examples of Mendelian syndromes that have heart defect include Noonan syndrome (the affected gene is PTPN11), Holt-Oram syndrome (the affected gene is TBX5), Marfan’s syndrome (the affected gene is FBLN). Epidemiologic studies show that genetic factors are the predominant reason of CHD although environmental factors may be relevant. A series of structural genes, signaling molecules and transcriptional factors have been found to participate in cardiac differentiation and structure formation. Illustrating the molecular mechanisms of heart development can help us understand the pathogenesis and provide theoretical basis for further treatment and management of CHD.Copy number variants(CNVs) are a form of structural chromosomal variation that manifest as deletions or duplications in the genome. The size of deletion or duplication of chromosomal segments ranges from 1Kb to several Mb often containing multiple contiguous genes. CNVs are the common reasones to cause chromosomal microdeletion or mocroduplication syndromes. There are over 100 different kinds of chromosomal microdeletion or mocroduplication syndromes which have been reported and over 40 of these syndromes have turned out to cause CHD. Based on these CNVs, many genes related to heart development have been identified which includes NKX2.5,ETS-1,ELN,TBX1 and so on. Some CNVs can lead to syndromic CHD such as Digeorge syndrome and Williams syndrome. While others can cause non-syndromic CHD. It is estimated that about ten percent of CHD have rare de novo CNVs. Further studies on these CNVs may provide us with new pathogenic clues of CHD.Classical G-banding chromosomal karyotyping is the gold standard of analyzing chromosomal anomalies. But some structural anomalies less than 4-10Mb are usually been missed by G-Banding karyotyping. The detection rate of chromosomal abnormalities depends on banding quality and the experiences of technologists themselves. With the rapid development of molecular technologies, more and more molecular diagnostic methods such as FISH, MLPA, array-CGH, SNP-array have been applied clinically. Espically with the mature of Chromosomal Microarray Analysis(CMA), we can scan the whole genome in one experiment and have a high resolution. Because of some disadvantages like high expensive, strict standards of laboratory and short of qualified technologists, CMA have not been widely used in China. While MLPA has been widely used in many laboratories because of its low cost, easy operation and short experimental period. But MLPA is a probe targated method, and some chromosomal anomalies would be missed beyond probe targated segments.By collecting samples(amniotic fluid or umbilical cord) from fetuses with congenital heart malformations as our research object, we can overcome some shortages of current researches about CHD, such as missing rare types of CHD when selecting living cases with CHD. We hope that our research can detect new genetic variantions that cause CHD in fetal stage and help us understand comprehensively the pathogenesis of CHD. By comparing three different genetic technologies used in this research(chromosomal karyotyping, MLPA and CMA),we can understand their advantages and disadvantages.Method:(1):Collecting the amniotic fluid samples or umbilical cord samples from fetuses with CHD during the second trimester of pregnancy.(2):Cells from amniotic fluid samples were cultured to carry out G-banding karyotype. DNA were extracted from Cells of amniotic fluid samples to conduct MLPA. The aim of MLPA of kit P095 is to screen aneuploids of chromosomal 21,18,13,X,Y, The aim of MLPA of kits P036 and P070 is to screen subtelomeric deletion or duplication. The aim of MLPA of kit P245 is to screen 21 common microdeletion syndromes. Samples without aneuploid anomalies screened by G-banding karyotype and MLPA were further to carry out array-CGH or SNP-array analysis. The testing process of umbilical cord samples was same as that for amniotic fluid samples except that the umbilical cord samples did not take chromosomal karyotyping analysis.Result:31 anomalies were identified in 107 samples, including 22 pathogenic variations and 9 variants of unknown significance. The rate of total anomalies was 29%(31/107) and the rate of pathogenic variations was 20.6%(22/107). Among 73 isolated CHD samples there were 16 anomalies including 11 pathogenic variations and 5 variants of unknown significance. The rate of pathogenic variations was 15.1%(11/73). Among 34 CHD with eatracardiac abnormal samples,15 cases were found to have chromosomal anomalies, namely 11 pathogenic variations(32.4%,11/34) and 4 variant of unknown significance (VUS).Chromosomal karyotyping analysis was conducted on 72 samples and 14 chromosomal anomalies were found with a 19.4%(14/72) abnormal rate including 13 pathogenic variants and 1 variant of unknown significance. Total 107 samples were screened by MLPA and rate of anomaly was 20.6%(22/107), anomalies including 21 pathogenic variants and 1 variant of unknown significance.93 samples were screened by CMA and 20 anomalies had been found including 12 pathogenic variants and 8 variants of unknown significance. Among the 85 samples that showed no anomalies screened by G-banding karyotype and MLPA,9 samples were identified by CMA to have anomalies, including 1 pathogenic variant and 8 variants of unknown significance(VUS). Among the 20 anomalies identified by CMA,11 pathogenic variations(11out ofl2) and 1 variants of unknown significance(1 out of 8) were found by MLPA.The total 22 pathogenic variations included 3 cases of Down syndrome,3 cases of Edwards syndrome,2 cases of Patau syndrome,1 case of mosaic Turner syndrome,3 cases of interchromosomal unbalanced translocations,5 cases of 22q11.2 deletion syndrome,4 cases of Jacobsen syndrome and lcase of 8p23.1 microduplication. In this research 9 variants of unknown significance were detected with a rate of 8.4%(9/107). The abnomal chromosomal segment includes 3q13.13q13.2,5q12.1,14q21.2q21.3,15q13.3,18p11.31p11.22,18p11.31, Xq27.2, Xp11.3p11.23,7q. Among the 9 cases with variants of unknown significance,5 cases had isolated CHD and 4 cases had extracardiac anomalies.Conclusion:1.31 samples were identified to have chromosomal numerical or structural anomalies in 107 CHD samples with a 29% abnormal rate.22q 11.2 deletion and Jacobsen syndrome are the most common chromosomal abnoemalities.2. CNV is one of the common pathogenic factor that causes CHD. The rate of VUS was 8.4%, suggesting that VUS may play an important role in the development of heart malformations.3. MLPA is an economic and rapid method to investigate chromosomal anomalies in fetuses with CHD. In some medical institutions that can not carry out CMA analysis, MLPA is possible to be an option used to discover chromosomal anomalies.