Study On Noninvasive Screening Biomarker Of Trisomy 21 Based On Urine Proteomics

Trisomy 21,also known as Down syndrome,is the most frequently occurring birth defect in humans caused by trisomy of all or part of human chromosome 21,with a prevalence rate of 0.1 to 0.2% for newborn infants,characterized by mental retardation and dysfunction involving multiple systems throughout the body,especially the cardiovascular,neurological,and musculoskeletal systems.Owing to the increasing work pressure and maternal age,as well as the application of assisted reproductive technologies,the incidence rate of trisomy 21 has been rising.At present,there is no effective cure for trisomy 21,and both countries and families must spend a lot of manpower and material resources.Therefore,prenatal screening is very important for the early detection and intervention of trisomy 21.Maternal age and fetal neck translucency ultrasonography are commonly used clinical screening methods,combined with a variety of early-and mid-pregnancy serum prediction markers.The common clinical serum markers include human chorionic gonadotropin beta subunit,pregnancy-related plasma protein A,alpha-fetoprotein,and unconjugated estriol.When these prenatal screening tests predict that the fetus is at high risk for trisomy 21,the intervention of more invasive prenatal diagnostic tests,such as amniocentesis or chorionic villus sampling are required to determine the fetal karyotype.But invasive diagnostic tests carry a higher risk of miscarriage.Recent advances in genomics have led to the development of noninvasive prenatal screening test by sequencing the cell-free fetal DNA isolated from the maternal blood sample.Noninvasive prenatal screening has the advantages of high sensitivity（99%）and low false positive rate（1%）,but the long testing cycle,high test failure rate and high cost limit its wide application in clinic.As one of the common clinical test samples,urine could be collected noninvasively,rapidly,and in large-scale.In healthy individuals,70% of the urine proteins/peptides originate from the kidney and the urinary tract,and 30% are from the ultrafiltration of blood within the glomerulus.In normal urine,a total of 6,085 proteins have been identified.The tissues associated with these proteins are distributed in various organs of the body.The tissue with maximum numbers of highly expressed proteins detected in urine protein was brain,followed by the colon and stomach.Hence,urine may not only contain biological information from the urogenital system but also other distant organs and even the whole body.Studies have shown that urine peptidome and metabonome profiles of pregnant women with trisomy 21 fetuses are significantly different from those with chromosomally normal fetuses.Urinary proteomics offers a new research platform for noninvasive diagnosis in this context.We report here new biomarker candidates found by urinary proteomics profiling.Specifically,we used liquid chromatography-tandem mass spectrometry（LC-MS/MS）to analyze the proteomes of urine samples from 19 pregnant women（aged 28～44 years）carrying fetuses with trisomy 21 and 22 healthy pregnant women（aged 27～42 years）carrying fetuses with normal karyotype.We identified 53 differentially expressed proteins between the trisomy 21 group and the healthy group,and most of these proteins were associated with embryonic development.Importantly,tissue inhibitor of metalloproteinases 2（TIMP2）and lysosomal-associated membrane protein 2（LAMP2）were further selected as potential urinary protein biomarkers.The combination of TIMP2 and LAMP2 could differentiate fetuses with trisomy 21 from healthy controls with a sensitivity of 74%,a specificity of 82%,and an area under the receiver operating characteristic（ROC）curves value of 0.82（95% confidence interval,0.69 to 0.95）.Furthermore,the combination of urine TIMP2 and LAMP2 was able to distinguish trisomy 21 from healthy controls in the narrowed gestational age of 19 to 20 weeks（AUC1,95% CI 1 to 1）.As potential urine noninvasive screening candidate markers for trisomy21,these two proteins warrant further clinical research in larger study samples.The differentially expressed proteins identified in the maternal urine will help us further understand the pathogenesis of trisomy 21.The combination of two candidate biomarkers,TIMP2 and LAMP2,may provide a potential non-invasive method for screening of trisomy 21.