Establishment Of Cell-based Non-invasive Prenatal Testing And Its Application On The Diagnosis Of Monogenic Diseases

Objective: Non-invasive prenatal testing(NIPT)for aneuploidies that using cell-free fetal DNA(cffDNA)in maternal peripheral blood is widely used in clinical practice.However,cffDNA does not contain sufficient fetal genetic information to diagnose monogenic diseases.Peripheral blood of pregnant women contains few fetal nucleated red blood cells(fNRBCs).In this study,a new non-invasive approach for the capture of fNRBCs for monogenic disease diagnosis was developed.Methods: Firstly,in this study,lymphocytes in peripheral blood were used as materials to verify the feasibility of single cell capture by laser capture microdissection(LCM),and single cell whole genome amplification(WGA)was performed by multiple annealing and loopingbased amplification cycles(MALBAC)to explore the opitimal exprimental conditions.Peripheral blood samples were collected from 24 pregnant women with a male fetuse for nucleated red blood cells(NRBCs)captured by LCM.The existence of fetal NRBCs(fNRBCs)were confirmed by amplification of SRY gene,and the success rate of this method was calculated.Then,genetic counseling was evaluated in12 pregnant women with family history of monogenic diseases including 5 familise with duchenne muscular dystrophy(DMD),3 familise with spinal muscular atrophy(SMA),2familise of congenital deafness,a family with duane radial ray syndrome(DRRS)and a family with early onset epilepsy encephalopathy(EOEE).After identifying the genetic causes of probands,fNRBCs captured via LCM were detected by Sanger sequencing or multiplex ligation dependent probe amplification(MLPA)and the results were compared with ones of amniotic fluids.An LCM-NIPT technique based on MALBAC was established.Results:(1)fNRBCs were detected in peripheral blood samples from 9 of 24 pregnant women with an accuracy of 37.5%(9/24).(2)Using the LCM-NIPT technique,fNRBCs were detected in 7 of 12 pregnant women with family history of monogenic diseases.Sanger sequencing was performed on 5 fNRBCs and prenatal diagnosises in 4 familise by fNRBCs succeeded,which included 2 familiese with DMD,a family with DRRS,and a family with EOEE.Sanger sequencing on an fNRBC from the family of congenitaldeafness failed due to allele drop-out(ADO)during amplification and MLPA assay failed to detect fNRBCs from 2 families with SMA.Conclusions: fNRBCs obtained via LCM can be amplifed by MALBAC and Sanger sequencing on fNRBCs can detect point mutations in monogenic diseases.Hence,LCM-captured fNRBCs have relatively good prospects for non-invasive prenatal screening and diagnosis.