University of Utah
Research foundation
Proposal Cover Sheet for
Funding Incentive Seed Grant
Technology Commercialization Project – New
Technology Commercialization Project – Renewal
1. Principal Investigator:
X
Luming Zhou
Department:
Pathology
Phone:
Email:
Fax:
587-4854
luming.zhou@path.utah.edu
581-4517
Co-Investigators:
Carl Wittwer
Bob Palais
David Eccles
Department:
Pathology
2. Title of Proposed Project:
HLA Matching for Transplantation by High Resolution Melting
Key Words (Research):
Key Words (Technique):
HLA, Genome matching
PCR, High resolution melting
3. Amount Request:
$35,000 X 2 years
4. Project period:
Start Date: 7/1/2006
End Date:
6/30/2008
5. Is this application a resubmission of a previously unfunded
Funding Incentive Seed Grant Program? Yes:
Technology Commercialization Project? Yes:
No:
No:
X
X
If yes, provide the date and common name of proposal
Date:
Common Name:
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6. Provide a concise abstract in the space provided below (no more the ½ page)
Abstract:
HLA genotype histocompatibility is an essential component of transplantation matching.
While in many cases, a matter of hours can mean the difference between life and death,
the current technique for HLA matching is an expensive and time-consuming process that
requires phenotypic allele typing or sequencing of at least the HLA-A, HLA-B, HLA-C
and HLA-DR loci. We are developing an alternative high-resolution melting technique
to determine the HLA identity among family members that is fast (< 1 hour) and
inexpensive. In preliminary studies, the melting analysis was performed on highresolution melting instruments in combination with a special double strand DNA dye.
Distinct melting curves immediately rule out any possibility of compatibility and need for
further typing, while identical melting curves suggest the same genotype. In this project,
we hope to carry this method forward to where melting curve identity becomes a
sufficient as well as necessary condition for determining HLA identity and
transplantation compatibility. Homogeneous genotyping with high resolution melting is a
very cost-effective and rapid method that could replace conventional HLA typing for
assessing transplant compatibility. The potential market for this new technique in the
diagnostic laboratory setting is substantial.
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Technical Proposal
General Background. Human Leukocyte Antigens (HLA) are cell surface proteins of
white blood cells and other tissues of the body which play a key role in transplant
tolerance or rejection. Matching between donor and recipient is advantageous. HLA
proteins form two major groups: class I (HLA-A, -B and -C), class II (HLA-DR, -DQ).
The currently accepted techniques for determining the HLA type include serotyping with
specific antibody reagents, allele specific PCR, hybridization with nucleic acid probes
and direct sequencing of the HLA genes. Because a large number of loci need to be
tested, the cost is about $650 to $2000 per patient. Complete genotyping of HLA is
necessary when donor and recipient are unrelated. However there is a 25% chance of a
perfect HLA match between siblings and for this reason organ transplant between
siblings is preferred when HLA matches occur. In this case it is not necessary to identify
their HLA alleles, only to they are the same. The technology for determining the melting
behavior of DNA has improved dramatically in the last few years (1,2) and it is now
possible to use fluorescent nucleic acid-binding dyes to generate highly accurate and
reproducible melting curves of PCR amplified gene fragments (3). With these assays one
can easily distinguish heteroduplexes from homoduplexes and in some cases distinguish
homoduplexes that differ by one or a few base changes. We propose to apply this
technology to the problem of matching HLA allele types between siblings. HLA
matching by high-resolution melting is rapid and inexpensive and appears to have utility
in compatibility testing, especially within families considering living related transplants.
Not only is it cost-saving, more importantly it can be life-saving in many cases where
time is of the essence.
Commercial Application of the Research.
Market size. The market for HLA matching is substantial. The Organ Procurement
Transplantation Network (http://www.optn.org) and the Center for International Blood
and Marrow Transplant Research (http://www.cibmtr.org), report there were 28,109 solid
organ and about 30,000 bone marrow transplants performed in 2003 in the United States.
Testing of each recipient plus (on average) two potential donors for HLA matching
means about 84,327 persons need to be tested for solid organ transplants and 90,000
persons need to be tested for bone marrow transplants. About 10% of solid organ
transplants and 65% to 80% of bone marrow transplants are between siblings.
Comparison with currently available products. . For solid organ transplants, it is
necessary to test at the antigen level for HLA-A, -B, -DR and -DQ. Medium resolution
molecular methods such as Sequence Specific Priming (SSP) or Sequence Specific
Oligonucleotides (SSO) (4) which cost about $650 per person are currently required. For
bone marrow transplants, allele testing of HLA-A, -B, -C, -DR, and -DQ using high
resolution methods is needed. The highest resolution is HLA sequencing of amplified
DNA and costs $2,000 per person. All these methods take about two weeks. In contrast,
the method we propose costs just $5 per person (reagents and consumables) and takes
approximately 30 minutes. High-resolution melting analysis with the double strand DNA
(ds/DNA) dye LCGreen I was recently introduced as a closed-tube method for
genotyping (3). This technique has been applied to SNP typing (5), unlabeled probe
genotyping (6), and mutation scanning for the MCAD (7), c-kit (8), and SLC22A5 genes
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(9). High-resolution melting of PCR products with DNA LCGreen I can identify a single
heterozygous base pair in amplicons as large as 1000 bp (10). In highly polymorphic
regions, different genotypes usually have different melting curves, but there is some risk
that distinct genotypes have nearly identical melting curves. In this case, 1:1 mixing of
samples produces heteroduplexes that are easily detected by high-resolution melting
curve analysis. (Figure 1).
Figure 1. HLA-A genotypes 0101/0101 and 3101/3201 have similar melting curves but
the 1:1 mixed melting curve is clearly shifted.
0101/0101+3101/3201
0101/0101
3101/3201
We recently performed HLA-A matching by high-resolution melting on the large
CEPH family UT1331 (11). There are 17 members in three generations in this family
(Figure 2a). Nested PCR (two-steps with dilution of product between steps) was
performed. Figure 2b shows the melting curves of HLA-A exon 2 PCR products
amplified from 17 members of this CEPH family. The curves clustered into six different
melting curve groups, indicating six different HLA-A genotypes in the family. When
HLA-B, -C and -DRB1 were tested in 11 siblings, the same siblings were consistently
grouped together. This result was confirmed by sequence-base typing. The melting
analysis requires only 1-2 min after PCR, and rapid PCR is typically performed in 15-30
min. Combining amplification and melting analysis in a closed-tube system routinely
requires less than an hour. In contrast to current molecular or serologic typing, highresolution melting is a simple, rapid, inexpensive method to establish HLA identity
between siblings.
Figure 2a, 2b . CEPH family UT1331 Pedigree and Melting Curves of HLA-A exon 2.
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Patentability. A patent application US-20060019253 has recently been published
(Amplicon melting analysis with saturation dyes), that is co-assigned to the University of
Utah and Idaho Technology. This application covers some aspects of HLA matching by
amplicon melting (generic method and dsDNA composition), but is limited in its scope.
In addition, we have two additional invention disclosures that have not yet been licensed.
These are U-3838 (Multilocus amplification for HLA matching by melting analysis), and
U-3703 (Automatic clustering and classification of homozygotes and heterozygotes by
high-resolution melting curve similarity). Both of these disclosures focus on practical
implementation of the HLA matching technology. Furthermore, we anticipate that
additional patentable inventions will arise from the work currently proposed.
Specific goals, objectives, project milestones and anticipated results. We will develop
a one-step, homogeneous procedure for HLA matching by high-resolution melting that
can replace conventional HLA typing for assessing transplant compatibility. The method
will be validated by a blinded comparison against current HLA typing practice after
sample de-identification.
Year 1 milestones: Develop a one-step (non-nested) PCR method for HLA
matching by high-resolution amplicon melting without opening the PCR tube. All
necessary HLA genes will be amplified under the same conditions on the same plate.
Automatic clustering algorithms for grouping identical HLA types will be developed.
Finally, the technique will be demonstrated using DNA from three extended CEPH
families.
Year 2 milestones: Validate the method by a blinded comparison against
conventional HLA typing. This study will include twenty clinical families that have had
a living-related transplant. We anticipate no errors.
Description of project plan including anticipated barriers and technical difficulties.
Because the members of the HLA class I gene family (HLA-A, B, C, plus several pseudo
genes) share extensive sequence similarity, a nested (two-step) PCR strategy has been
used in the past to amplify selected exons of each of the HLA-A, B and C genes. Because
of the highly polymorphic nature of the HLA region, finding adequate primers for singlestep amplification is a challenge. The most attractive solutions are:
1) Amplify exon 2 and exon 3 together in a multiplex PCR for each Class I HLA
locus (A, B, and C). Three PCRs would be required. This method has been used in
proprietary commercial kits.
2) Find consensus primers that amplify one exon (either 2 or 3) of all HLA Class I
loci (HLA A, B and C). Two PCRs would be required.
The most successful method will be tested against CEPH familys UT1331,
UT1355, and UT1413.
For year 2 we will test HLA “matching by melting” on a large number of clinical
samples that have been de-identified and typed by either serology or sequencing by the
University of Utah HLA laboratory under the direction of Dr. David Eccles
(coinvestigator). We have an approved IRB (#12977) to access DNA from up to 100
families’. High-resolution melting instruments are available in the investigator’s
laboratory, including single capillary tube, and 96 well plate instruments.
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References
1. Talkemoto SK, Terasaki PI, Gjertson DW, Cecka JM. Twelve years’ experience
with national organ sharing of HLA-matched cadaveric kidney for transplantation.
N Engl J Med 2000: 343: 1078.
2. Opelz G. Collaborative Transplant study. University of Heidelberg, Germany
(http://www.ctstransplant.org).
3. Wittwer CT, Reed GH, Gundry CN, Vandersteen JG, Pryor RJ. High-resolution
genotyping by amplicon melting analysis using LCGreen. Clin Chem 2003: 49:
853-60.
4. Helmberg W. Storage and utilization of HLA genomic data--new
approaches to HLA typing. Rev Immunogenet 2000; 2(4): 468-76.
5. Lie M, Pror R, Palais R, Meadows C, Erali M, Lyon E, Wittwer CT. Genotyping
of Single-Nucleotide Polymorphisms by High-Resolution Melting of Small
Amplicons. Clin. Chem 2004; 50: 1156 - 64.
6. Zhou L, Myers AN., Vandersteen JG, Wang L, Wittwer CT. Closed-Tube
Genotyping with Unlabeled Oligonucleotide Probes and a Saturating DNA Dye.
Clin. Chem 2004; 50: 1328 - 1335.
7. Magera MJ, Gunawardena ND, Hahn SH, Tortorelli S, Mitchell GA, Goodman
SI, Rinaldo P, Matern D. Quantitative determination of succinylacetone in dried
blood spots for newborn screening of tyrosinemia type I. Mol Genet Metab. 2006
Jan 28: 112-120.
8. Willmore C, Holden JA, Zhou L, Tripp S, Wittwer CT, Layfield LJ. Detection of
c-kit-activating mutations in gastrointestinal stromal tumors by high-resolution
amplicon melting analysis. Am J Clin Pathol. 2004 Aug;122(2):206-16.
9. Dobrowolski SF, McKinney JT, Amat di San Filippo C, Giak Sim K, Wilcken B,
Longo N. Validation of dye-binding/high-resolution thermal denaturation for the
identification of mutations in the SLC22A5 gene. Hum Mutat. 2005 Mar; 25(3):
306-13
10. Reed GH, Wittwer CT. Sensitivity and specificity of single-nucleotide
polymorphism scanning by high-resolution melting analysis. Clin Chem. 2004
Oct; 50(10):1748-54.
11. Zhou L, Vandersteen J, Wang L, Fuller T, Taylor M, Palais B, Wittwer CT. Highresolution DNA melting curve analysis to establish HLA genotypic identity.
Tissue Antigens. 2004 Aug; 64(2):156-64.
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