Creating a Targeted Next Generation Sequencing Assay to Identify RH antigens from DNA for Stem Cell Transplants

Abigail Joseph

Principal Investigator: William Lane MD, PhD

Mentors: Peter Tonellato PhD; Helen Mah, MS; John Baronas

Dana-Farber Cancer Institute

Using DNA assays to determine human leukocyte antigen (HLA) type is standard practice when seeking to find stem cell transplant donor matches. Recently, next generation sequencing (NGS) has been successfully developed to perform accurate HLA typing. As a result, stem cell donors are typed using DNA samples collected with cheek swab kits. ABO and Rh mismatches between donor and recipient can result in major and minor incompatibilities that can delay erythroid engraftment and require multiple rounds of red blood cell (RBC) transfusions. An ABO and Rh NGS assay could improve the stem cell transplant matching process because only DNA samples are collected from prospective donors. However, one major obstacle in using NGS to determine the presence of Rh antigens from DNA is the similarity between the RHD and RHCE genes. The objective of our experiment was to determine whether a targeted NGS assay could be developed to determine the specific Rh antigens in a patient’s RBCs. We performed long range PCRs to test optimal primer combinations for successful amplification of the genes. Gene specific indexes were added to the RHD and RHCE PCR products, prior to sequencing, so identical regions of the genes could be clearly labeled. The results of the sequence alignments showed that the primers that targeted RHD exons 1-6 and RHCE exons 1-4 resulted in the largest PCR products while maintaining strong alignment coverage. In addition, the DNA reads aligned according to our expectations allowing us to predict the patient’s antigen type as D antigen positive, homozygous for the C antigen, and homozygous for the e antigen. These findings suggest that Rh antigens can be determined from DNA assays without serologic testing. Consequently, the same DNA sample used for HLA typing can be used for Rh antigen typing, thus streamlining the stem cell donor match process.

Currently HLA (human Leukocyte Antigen) typing is done completely from DNA

● However, RBC antigens still cannot be determined from DNA and must be confirmed

serologically

● This hinders the process of finding donors for Stem Cell Transplant,

○ Although a DNA sample, such as spit or cheek cells, that are sent in can be used

to determine a potential donor's HLA type, that same sample can’t be used to

determine RBC type

○ Donor must physically come in to donate blood and test must be done

serologically, which is time consuming and expensive

● This is especially relevant for patients with leukemia or Lymphoma who need Stem Cell

Transplants since their immune systems were eliminated in the treatment process

● There is a need to find exact matches or else the patient is at risk for HTR (hemolytic

Transfusion Reaction) or GvHD (Graft vs. Host Disease)

● Purpose is to create a targeted assay that uses Next Generation Sequencing to confirm

RH, RhD and RhCE, antigens on RBC similar to how HLAs are confirmed

● Then theoretically one DNA sample could be used to find the most promising exact HLA

and RBC antigen matches, so only a few would have to be brought in for serologically

confirmation of the DNA results.

● Problem with current attempts to sequence the RhD and RhCE is that they are very

similar gene and standard methods of hybrid capture and amplicon sequencing do not

work

○ Some parts of RhD and RhCE are so similar so sequencing without long enoug

reads can fail to identify which DNA come from RhD and which from RhCE

● The approach we used is very long range PCR

○ It can create gene specific sequences, which circumvents the problem that the

genes look very similar

○ By allowing us to add gene specific tags so we can know which gene it came

from when sequencing

● First step is find a primer that would amplify the Rhd gene DNA

○ Looked at PCR products to determine size and quantity

● Next the goal is to sequence the DNA, using an illumina miseq,

● Take sequencing data dn see which genes they align to

● Last is to examine the sequencing data using a software to determine whether the

sequencing allows us to conclude that the someone has an RhD antigen or if the

sequencing is inconclusive

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3926707/

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http://path.upmc.edu/cases/case101/images/gross5.jpg

Presentation

I. Intro A. What are the consequences of not matching type when giving stem cell

transplants 1. Stem Cell transplants are typically given to leukemia and lymphoma

cancer patients, who have had their immune systems destroyed by treatment and need the transplants to replenish their immune system

2. GvHD (graft vs. Host Disease) a) Diseases can be fatal

3. HDFN or HTR for RBC B. How can DNA help provide more accurate blood typing?

1. For the past 5 yrs HLA (human leukocyte antigens) aka white blood cell antigens have been typed completely from DNA

2. No longer needs to be done serologically, time consuming process, prone to human error, expensive lab procedures and reagents

3. However RBC (red blood cell) typing still cannot be confirmed from a DNA sample but must be tested serologically

C. Our question 1. We chose to look at the RhD and RhCE antigens, to determine whether

we could create a targeted NGS assay to reliably determine the D, C, c, E, e antigens present on a patient's RBC from a DNA smaple

a) Explain what D anitgen is, determine (+) or (-) blood type 2. These antigens are especially problematic to determine with DNA

sequencing because they are very similar to each other, they can even containing completely identical exons, and often sequences from one gene can align to another, for accurate typing it is essential that reads (sequences) align to the appropriate gene

II. Set Up - ask for Illumina Prep procedure A. Before we could sequence the RhD and RhCE genes we had to determine which

primers would give us the highest concentration of the genes and the most accurate “cuts”

1. We focused only on the first 7 exons in both - WHY B. Performed 12 very long range PCR for each RhD and RhCE with different

primers, and used a gel to determine how much volume was produced, and a standard to approximate the length of the DNA strands

1. Show image of PCR explain how each well increases in size as it contains more exons

C. Before putting the the DNA in the Illumina miseq, we tagged the DNA that was Rhd and that was RhCE, with different tags, so at the end of the process we know which DNA came from where

1. How does the tagging work???

D. The Illumina miseq take about 22 hrs to sequence the gene and then we download the data into IGV (Integrative Genomics Viewer) to analyze the success of each of the different primer set combinations

III. Data and Interpretation A. Show the what the RhD and RhCE look like on one of the most successful trials

and one of the mediocre trials 1. Explain how to tell whether someone has high or low coverage 2. Maybe how to determine their genotype from the one base pair 3. Explain why there might be low coverage

a) Misalignment - graphic from Dr. Lanes Slide IV. Context and Importance and Future Directions

A. Only 1 of the more than 300 RBC antigens, however it's the most important antigen after the ABO group

B. When searching for Donors, in the future one sample of spit or cheek swab would allow a doctor to determine your entire specific blood type,