Study Of Genetic Etiology Of Whole Exome Sequencing In Fetus With Ultrasound Structural Abnormal And In Family With Rare Disease

Background:With more than 1.4 billion people,China is the country with the highest incidence of birth defects.According to the "China Birth Defects Prevention Report(2012)",the current incidence of birth defects in China is about 5.6% of births,with up to 900,000 cases each year."Birth defect" refers to an abnormality of structure,function,or body metabolism at birth resulting in physical or mental disabilities or death.Birth defects not only seriously endanger the survival and quality of life of children,and affect the happiness and harmony of the family,but also causes huge potential life loss and social and economic burdens.Birth defects have become a public health problem,and it is urgent to take appropriate intervention measures to prevent it.Birth defects can be caused by genetic factors such as chromosomal aberrations and gene mutations,or environmental factors,as well as the interaction of these two factors or other unknown reasons.Among them,abnormal genetic factors are an important cause of birth defects.So identifying genetic alterations associated with birth defects is essential in birth defects prevention.High throughput DNA sequencing methodology(next generation sequencing;NGS)has rapidly evolved over the past decade.Compared with the Sanger sequencing,NGS not only increases the sequencing throughput,but also greatly reduces the sequencing cost.The combine of DNA target capture and high-throughput sequencing can efficiently sequence the protein-coding region of the human genome,which is called "whole exome sequencing(WES)".Now,WES has become a powerful tool that can identify pathogenic variants related to the patient’s phenotype,in which conventional methods fail.Diagnostic ultrasound is the most wildly and commonly used to detect birth defects during pregnancy.It has been shown that 65.9% fetal abnormalities detected by ultrasound were structural abnormalities.It is known that fetal structural abnormalities are often related to genetic mutations.Due to the limitation in resolution,traditional karyotype analysis,FISH and CMA technology can not identify all the genetic alterations in fetuses with structural abnormalities.It is urgent required a new approach to determine genetic abnormalities resulting in structural abnormalities in prenatal diagnosis.Therefore,rational use of WES to find pathogenic variants of fetal structural abnormalities,would avoid the birth of children with structural deformities and reduce birth defects.For probands in families with rare diseases,WES can also be used to for molecular diagnosis,which facilitates the identification of disease types and genetic causes.The results may provide guidance for the family’s reproductive needs and prevent the recurrence of birth defects.Method:(1)Use invasive methods(Amniocentesis or Cordocentesis)to obtain intact fetal cells,and perform karyotyping and CMA after culture.(2)Extract DNA from fetal amniotic fluid cells with abnormal ultrasound structure to complete fetal WES;if trio-WES is used,fetal parent’s DNA must also be extracted.(3)Report the mutations related to the abnormal structure of the fetal ultrasound,integrate the fetal phenotype and family status,the Sanger sequencing verification results,existing reports and research,and interpret the variants according to the ACMG guidelines.(4)Whole exome sequencing analysis of probands with suspected rare diseases,minigene verifies the pathogenicity of variants.Result:(1)The 119 fetuses with abnormal ultrasound structures included in the study failed to clarify the etiology through karyotype and CMA.(2)Whole-exome sequencing the average sequencing depth of 119 fetal samples was129.91±28.93×,and the average coverage was 99.84%,of which the average coverage was98.91% greater than 20×.(3)119 cases of fetuses with abnormal karyotype and ultrasound structure that failed to be detected by CMA were further detected by WES and 17 cases(17/119,14%)were found to be of diagnostic significance,and the conclusion of 26 cases was not clear yet(26/119,22%),76 cases were still negative(76/119,64%).(4)Sequencing of a suspected rare disease proband found a compound heterozygous mutation in the COL7A1 gene.The minigene experiment results showed that the mutation c.5532+4＿5532+5del AG can cause the skipping of exon 64 of the COL7A1 gene,forming abnormally spliced transcripts and with another mutation of this gene c.3867 del T,causes the RDEB disease phenotype of the proband.Conclusion:This study provides a reference for the application of WES in clinical practice.Molecular diagnosis may be obtained by WES analysis for fetuses whose karyotype and CMA analysis failed.WES combined with existing technology can prevent birth defects and guide reproductive;and it also can be applied to molecular diagnosis in rare diseases.WES can broaden the understanding of disease phenotype-genotype and discover new pathogenic genomic alterations,which has certain practical and scientific value.