Application Of CNV-seq Technique To Detect Cytomegalovirus In Amniotic Fluid

Background: Cytomegalovirus(CMV)infection is one of the most common congenital viral infections,which can lead to serious birth defects such as early miscarriage,premature birth,stillbirth,or abnormal fetal development and malformations.Therefore,early diagnosis and treatment of CMV infection is essential to reduce adverse pregnancy outcomes such as stillbirth or miscarriage,improve the prognosis or quality of life,and reduce birth defects.Low copy number variation of deep whole genome sequencing technology sequencing(CNV-seq)as a prenatal diagnosis technology,which can be used to screen for the etiology of genetic diseases and birth defects associated with chromosomal abnormalities or genetic mutations.However,the research of CNV-seq on pathogenic microorganisms such as cytomegalovirus(CMV)mainly focuses on the genomic analysis of pathogens in clinical samples.Purpose: In this study,we used CNV-seq technology combined with real-time PCR technology to qualitatively detect and quantify CMV in amniotic fluid samples.We then investigated the clinical application value of CNV-seq technology in the diagnosis of CMV infection.Method:.Collect 931 patients who visited the Prenatal Diagnosis Center of the First Affiliated Hospital of Hainan Medical University from January 2021 to June 2022 as the research subjects.Clinical data of the patients,including age,gender,gestational age,obstetric condition,laboratory examination results,imaging manifestations,amniotic fluid and peripheral blood collection,CNV-seq and fluorescence quantitative PCR detection results,were collected through Lian-zhong and Jia-he electronic medical record system,telephone follow-up,and other methods Retrospective analysis of the diagnosis and treatment process and follow-up prognosis.Result : 1)Three out of 931 patients were diagnosed with CMV infection through CNV-seq,with a CMV detection rate of 0.3%;2)Obstetric ultrasound screening showed abnormalities in all the three patients.In case 1,obstetric ultrasound indicated oligohydramnios,neonatal craniocerebral ultrasound indicated premature brain and cardiac ultrasound indicated unclosed foramina ovalis.In case 2,obstetric ultrasound showed that fetal nasal bone was not shown.In case 3,fetal cardiac structural abnormalities,ventricular septal defect,aortic straddle and pulmonary atresia were indicated by obstetric ultrasound ",considering complex malformations of congenital heart disease.3)The serum specific antibody results of the three patients indicated negative CMV-Ig M and positive CMV-Ig G.The urine CMV-DNA copies of the neonates in case 1 and 2 were 4.54×104 copies/ml and 4.8×104 copies/ml,respectively,but the neonates in case 2 did not test the urine CMV-DNA.4)Prenatal diagnosis was performed in all the three patients,and CNV-seq results indicated that no pathogenic or suspected pathogenic variation of aneuploidy,conformity chromosome linked inheritance or autosomal dominant inheritance mode was detected.QF-PCR was performed on the amniotic fluid samples of case 2 and case 3,and the results also indicated that no aneuploid aberrations were found on the 13,18,21,X and Y chromosomes,and the karyotype analysis was 46,XN.The results of whole exome sequencing of the genome of amniotic fluid samples from case 3 showed that the DYNC2H1 gene c.3782G>T,c.1506T>C compound heterozygous mutations were clinically unknown variants,related to brachy thoracic dysplasia with or without polydactyly syndrome type 3,which is autosomal recessive or bigenic recessive.5)CMV was detected and analyzed in all three patients with CNV-seq prenatal diagnosis of amniotic fluid samples,and the sequence number/total sequence number ratios of the three cases were 179/2048166(0.000087),15619/1815815(0.008602),and13227/2736964(0.004833),respectively.6)The results of CMV detected by CNV-seq in amniotic fluid were verified by fluorescence quantitative PCR.The viral load of case1 was 1227.76Copies/ml,that of case 2 was 11639.00Copies/ml and that of case 3 was13754.60Copies/ml.7)Three patients had no obvious clinical manifestations of CMV infection during pregnancy and did not receive symptomatic supportive treatment such as the antiviral therapy.Up to now,no clinical manifestations related to the nervous system of CMV infection have been found in the neonates of three patients.Conclusion: CNV-seq technology may have certain application value for the clinical diagnosis of CMV infection,and may provide the related clinical basis for the construction of a one-stop prenatal diagnosis platform for CMV infection.However,follow-up large-sample,multi-center clinical studies are needed.