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Linz Researchers Say 454 Sequencing Will Improve Tissue Matching for Transplants

Researchers at the Blood Center Linz in Austria are betting on second-generation sequencing technology to improve tissue matching between donors and bone marrow or stem-cell transplant patients.
The researchers, who acquired a 454 Genome Sequencer FLX in May, said they eventually want to develop a sequencing method for routine use in HLA typing, which they say would be one of the first applications of 454’s technology in medical care.
Research groups elsewhere are working on similar methods using other approaches, among them microarrays or Sanger sequencing, but the main advantage of 454’s platform is its speed and resolution, according to Christian Gabriel, medical director of the Blood Center Linz.
“It’s not cheaper, but it will save time and we can get results with better and higher resolution that we could never get anywhere else,” said Gabriel, whose center won a grant for a 454 system from the Austrian government last year. “Our idea is to [develop] this into some sort of method which we can use for patient care.”
The human leukocyte antigen, or HLA, system is a region on chromosome 6 that encodes a large number of highly diverse genes, many of which have functions in the immune system. The six major antigens encoded by HLA genes are HLA-A, -B, -C, -DR, -DP, and DQ.
In order to prevent transplant rejection, HLA types between donor and patient need to be matched as closely as possible. Right now, the Linz center provides HLA typing for approximately 500 bone-marrow and stem-cell transplant patients a year, according to Gabriel.
HLA typing usually involves PCR-based assays, which can result in ambiguities. These can sometimes be solved by Sanger sequencing, though “you cannot discriminate between ambiguities where you have, let’s say, overlapping SNPs,” according to Gabriel.
That is when the researchers have to resort to family studies, which involve HLA-typing of several family members of a patient. “This is very, very time-consuming,” he told In Sequence. “It takes approximately a month until you are through.”
The Linz researchers now want to use 454’s platform to develop a method that would eliminate the need for family studies, thus reducing the time required for HLA typing. This could be crucial for the outcome of the transplant, according to Gabriel: Some studies say a three-month delay of a transplant increases the risk to a similar level as having a single HLA mismatch.
“We thought if we use the FLX, then it will be possible to discriminate between different haplotypes,” he said.
In a three-stage, two-year research project, the researchers initially plan to HLA-type a handful of patients with known transplantation history, followed by validation studies in 30 to 40 patients.
Initially, Gabriel said he and his colleagues plan to sequence a number of exons from HLA loci in order to resolve ambiguities that could not be solved previously by Sanger sequencing.
Next, they want to sequence an entire HLA locus from several PCR-amplicons in the same patients, which would allow them to discover novel SNPs.
Finally, they plan to develop a method to sequence chromosome 6 in its entirety, which includes the HLA genes and a large number of other genes. This would allow them to determine the HLA haplotypes as well as SNPs between the HLA genes.
The biggest challenge for the project will be the data analysis, according to Gabriel. He said his group is already working with bioinformaticists at the University of Applied Sciences Upper Austria in Hagenberg to establish a compute cluster for the project.
“The problem with the 454 system is that the capacity you need for [computer] hardware in routine and daily business would be much higher than [what is] provided by Roche/454,” he said. “If I want to run this on a routine basis, I need a machine [that does everything] automatically — quality assurance, sequence reads, alignments — within [a few] hours.”
In addition to HLA typing, the researchers also plan to use the 454 platform in other projects centered around immunogenetics and bacteriology, for example to sequence genes of immunoglobulin receptors and to study microbial pathogens by metagenomics. “We are looking to establish new methods for medical applications,” Gabriel said.

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