1. Inves&ga&ng the Correla&on between Cell Free DNA and Transplant Rejec&on/Injury
Roel Marania1, 2, Juliane Liberto2, Tara Sigdel2, Minnie Sarwal2
1. Abraham Lincoln High School
2. Department of Surgery, University of California, San Francisco
The Kidney is an organ that filters waste and maintains
chemical balances within the body. When the organ does not
perform these roles effectively, a solution is to undergo organ
transplant. After a patient has gone through kidney transplant
(Figure 1), they are at risk for organ rejection as the transplant
organ from the donor is considered as a foreign object by the
recipient’s immune system. In addition, organ can fail by non-
immune causes such as viral infection and drug toxicity.
Currently, the best method for early detection of transplant
rejection is through invasive tissue biopsy(1). The pathological
evaluations of tissue biopsy are not optimal. An optimal and
non-invasive method of transplant rejection of transplanted
kidney is an unmet need. In this project, we aimed to discover
a non-invasive urine assay to assess whether a patient is at
the risk of rejection and needs to be sent for a biopsy for
further evaluation or if the patient is free of such a risk and do
not need a biopsy evaluation.
Cell free DNA (cfDNA) is circulating DNA found in the blood
(2), and urine. cfDNA enters these fluids through cell death
and apoptosis, and could be an indicator of graft rejection or
injury. We hypothesized that cfDNA in the urine can be
quantified and there is a relationship between high copy
numbers of cfDNA and acute rejection/graft injury.
Investigating the correlation between rejection cases and
cfDNA can enable physicians to effectively detect early stages
of organ rejection to help prolong the age of the transplanted
organ.
References
1. Sigdel, Tara K., Matthew J. Vitalone, Tim Q. Tran, Hong Dai, Szu-chuan Hsieh, Oscar Salvatierra, and
Minnie M. Sarwal. "A Rapid Noninvasive Assay for the Detection of Renal Transplant
Injury." Transplantation. U.S. National Library of Medicine, 2013. Web. 25 July 2016.
2. Bryzgunova, O. E., and P. P. Laktionov. "Extracellular Nucleic Acids in Urine: Sources, Structure,
Diagnostic Potential." Acta Naturae. A.I. Gordeyev, 2015. Web. 25 July 2016.
Ø We used urine samples collected from kidney transplant patients.
(n=74)
Ø Samples were centrifuged and supernatant was frozen at -20ºC until
ready for extraction.
Ø cfDNA was extracted with the QiAamp Circulating Nucleic Acid Kit
(Qiagen Cat No. 55114)
Ø Copy numbers were quantified using Quant Studio™ 3D Digital PCR
(Lifetech Cat No. 4481097)
Ø cfDNA was quantified using primers specific to a region on Chr1.
Ø Urine Creatinine was measured with the Quantichrom Creatinine Assay
Kit (Cat No. DICt-500)
Ø Optical Density was read on SpectraMax M Series PLUS-384, at OD
510.
Ø Data was normalized using the equation:
Our results conclude that there is a clear relationship between
high copy numbers of cfDNA and transplant rejection. This
indicates that cfDNA can act as a substitute biomarker for
transplant rejection and injury. This could facilitate physicians
to send patients for biopsy evaluation or not, because biopsy
is still the “gold standard” for transplant rejection detection.
This assay can also be applied to other organs such as the
heart, because cfDNA is found in the bloodstream as well as
plasma.
The study will have to be expanded to more urine samples as
well as including longitudinal samples to make the study more
robust and reliable. We believe that, once validated, this
assay can be used for clinical monitoring (Figure 3).
Introduc&on
Figure 2. Increased cfDNA in the AR and transplant injury urine. Mean
urinary Chr1 cfDNA in AR (8852.7 ±20484.8) was significantly greater
compared with no AR-Normal (135.1 ±335.6) or those with other injuries
(1526.8 ±2818.3).
Materials & Methods
Results
Copy number of cfDNA in the urine of AR patients was significantly higher
AR (8852.7±20484.8 copy/mg/urine creat.) than those without rejection
or injury (135.1 ±335.6 copy/mg/urine creat.) (p =0.003). cfDNA in the
urine collected from patients with no-AR but with other form of injury was
also significantly higher (1526.8 ±2818.3 copy/mg/urine creat.) compared
to non-AR with no injury (135.1 ±335.6 copy/mg/urine creat.) (p =0.003)
(Figure 2).
Conclusions
Future Directions
Acknowledgements:
• Sarwal Lab
• Mentors: Juliane Liberto, Izabella Damn, Szu-Chuan Hsieh
• PI: Minnie Sarwal, Tara Sigdel
• SEP Staff and Interns
• Nominating Teacher: Mrs. Julie Reis
Figure 3. Potential utility of cell free DNA (cfDNA) in urine.
Urine can be collected at time of patient’s clinic visits to monitor
cfDNA. A baseline cfDNA level for each patient is determined by
measuring cfDNA at a time when graft functions properly. Urine
collected at the time of visit (scheduled or due to suspected
dysfunction) are then compared to the patients’ individual
baseline. A substantial increase in cfDNA could be used for
clinical monitoring.
Figure 1. Diagram of a patient who has undergone a kidney
transplant. The new organ is grafted into the patient inferolateral to
the original damaged kidneys.
copy#
mL urine
/
mg urine creatinine
mL urine