The Application Of Noninvasive Diagnosis Of Cell-Free Dna In The Monitoring Of Lung Transplant Rejection

For patients with end-stage lung disease,lung transplantation can increase their life expectancy and improve their quality of life.Though immunosuppressant drugs have been widely used in patients having lung transplantation,acute rejection(AR)is still one of the major complications and highly prevalent in the first year post-operationally.However,there is no reliable serum marker available to monitor AR after lung transplantation.Transbronchial biopsy,the gold standard for diagnosis,is an invasive procedure which may cause side effects.Therefore,the easy and noninvasive approaches for early and accurate lung allograft rejection tests are in great need.It is possible to determine transplant rejection by detecting the level of ddcfDNA content after lung transplantation,which could be used as a biomarker with the development of NGS.Through simulation,we tested whether it is viable to extract the genome DNA and cfDNA from the donor and recipient and build the database.Then we used the Single Nucleotide Polymorphisms(SNPs)analysis method to distinguish ddcfDNA.After recruiting patients,we collected donor and recipient's peripheral blood for NGS and genotyping.The whole genome sequencing of post-transplant recipients was collected to detect the changes in proportion of ddcfDNA The results were compared to verify the accuracy of the method in monitoring transplant rejection.An effective method to extract and construct databases of donor and recipient's genome DNA and cfDNA was established.We have applied an innovative approach for the quantification of ddcfDNA based on mini-screen target capture array for genotyping which is 7.7 Mb total capture space including 56 Kb SNPs.The simulation experiments and clinical experiment showed that the chip can effectively distinguish the donor's SNPs.We have also developed an innovative way to calculate donor signal via whole genome sequencing of recipient plasma.A significant linear correlation between % Donor in library and % Donor DNA was shown(R~2=0.98).The proportion of ddcfDNA was significantly different between the rejection group(4.74±1.78%,mean ± SD)and the no rejection group(1.62±0.58%,mean ± SD),which was validated by biopsy with 41 clinical samples.According to these results,we may find that ddcfDNA levels from lung allograft recipients increased when rejection events occurred.The sensitivity of this method was 84.6% and the specificity was 100% This method can be applied monitor AR after lung transplantation.