Molecular Genetic Diagnosis For Fetus With Congenital Heart Disease

Congenital heart disease is the most common birth defects. About one third of the newborn suffering from congenital heart disease would be extremely harmful in one months after birth due to a serious of illness and malformations if intervention and treatment were not timely. Epidemiological studies have shown that environmental factors and genetic factors together lead to congenital heart disease in which genetic factors play a major role. Genetic testing of fetus with congenital heart fetal can make the genetic background clear,and provide important information for the genetic counseling and help assess the risk of re-birth couples; also has the guiding value of prognosis judgment of fetal with congenital heart disease and surgical effectNumerous studies confirm that genetic variations causing congenital heart Comprise chromosome mutation, genome disease, gene mutations,and so on.In this study, we use a combination of G-banding technique, multiplex ligation-dependent probe amplification(MLPA) technology, Single Nucleotide Polymorphisms array-based comparative genomic hybridization (SNP-array) and exon capture-high-throughput sequencing integrated technology systems sequentially to diagnose congenital heart disease geneticlly and to find genetic causes of congenital heart disease,and assess the risk of recurrence and to help determine the prognosis of fetus,and also to assess the effect of the system used in genetic testing of fetal with congenital heart disease.Part I Copy number variation detection for fetal with congenital heart diseaseObjective:To explore the role of MLPA technology and SNP-array technology in the detection of copy number variation of congenital heart disease in the fetus and evaluate their function in prenatal diagnosis.Methods:Two hundred and thirty-six fetus with congenital heart disease, who have nomal G-banding results, were included in this study. Using MLPA technique to detect 22q11.2 microdeletions and microduplication of genome.in these fetal. Two hundred and twenty-six fetus with negative results were detected by array SNP-technique to detect the whole genome copy number variations. We go on data analysis of the obtained fetal CNV results, and make clear genetic cause of congenital heart disease to conduct genetic counseling.Results:Ten cases with 22q11.2 region copy number variation including nine cases with microdeletion and one case with microduplication were detected in Two hundred and thirty-six cases of fetus. Of the nine fetus with 22q11.2 microdeletion,eight cases of microdeletion were de nove, one microdeletion and microduplication were derived from his father. SNP-array results found that in the remaining two hundred and twenty-six cases of fetus with congenital heart disease, after database comparison there were seventy one cases of fetus with CNV, including twenty-three cases of fetus with pathogenic CNV, forty cases of fetus with benign CNV, eight cases of fetus with unknown CNV,. the rate of pathogenic CNV was 10.1%(23/226). Combining MLPA technology with SNP-array technology for detecting the etiology of fetus with congenital heart disease can improve the positive rate of 14.0%(33/236) compared with conventional karyotype analysis.Conclusion:Genome copy number variation is one of the important causes of congenital heart disease. Combing MLPA with SNP-array can significantly improve the detection rate of CNV in patients with congenital heart disease,and it is helpful for prenatal counseling.Part II Detection of gene mutations of fetal with congenital heart disease-exon capture-high-throughput sequencing technologyObjective:To explore the possible pathogenic genetic evidence of congenital heart disease and to evaluate the value of exon-capture high-througput sequenc-ing technology in the molecular genetics of prenatal diagnosis.Methods:Forty-four fetus with congenital heart disease who have nomal G-banding and MLPA and SNP-arrayanalysis results, were included in this study. A customized capture microarray targeting sixty-three genes which were well-known CHD associated genes were made and captured DNA fragments were sequenced by high-throughput sequencing.Through base calling, database compa-rison, software analysis, we obtained the final pathologic mutations. Pathologi-cal mutations were validated using Sanger sequencing.Results:We got 3560 SNPs, Sixteen mutations are considered pathogenic where five mutations have been reported previously and the remaining eleven muta-tion have not been reported as far as we know. There are two frameshift muta-tions and fourteen missense mutations involving ten genes:CITED2, CHD7, N-OTCH2, JAG1, MYH7, ZFPM2, HAND2, MLL2, MYH6, MIDI. Sanger sequen-cing confirmed the sixteen mutations detected by exon capture-high-throughput sequencing, and revealed all of the sixteen mutations are de novo.Conclusion:In this study, using the customized exon-capture and sequencing technology we found five reported pathogenic mutations and eleven suspected pathological mutations in 44 fetus with CHD. Our study suggested that gene mutation is one of the important reasons of congenital heart disease, this techn-ology can significantly improve the improve the positive rate of detection of fetus with congenital heart disease and has better clinical value.