Box 1. Human leukocyte antigen genotyping.

A Stanford University team has developed a new human leukocyte antigen (HLA) sequencing method that could be more cost effective and accurate than existing technologies to extend the applications of HLA genotyping, which has previously focused on matching bone marrow donors and recipients.

HLA polymorphisms are clinically relevant because specific alleles have been associated with various autoimmune diseases including multiple sclerosis (MS), celiac disease, rheumatoid arthritis (RA) and type 1 diabetes.4-7 Additionally, matching HLA genotypes is essential for host acceptance of bone marrow transplants.8

Despite the clear importance of knowing a patient's HLA genotypes, the actual sequencing is expensive and time consuming. Most methods exclusively sequence the antigen-binding cleft because it is the most variable, but this can lead to undetected sequence variation in other regions of the HLA genes.

In a paper published in the Proceedings of the National Academy of Sciences, Stanford's Michael Mindrinos and colleagues developed a high throughput HLA sequencing method that maps most of the HLA genome to reduce or eliminate ambiguity.

The method also identified previously undetected HLA alleles that could be implicated in disease susceptibility or could further improve matching of patient and donor transplant compatibility.

Mindrinos is associate director of the Stanford Genome Technology Center. The paper also included researchers from the university and the Howard Hughes Medical Institute.

The team developed primers to sequence the major histocompatibility complex class I A (HLA-A), HLA-B, HLA-C and major histocompatibility complex class II DR b1 (HLA-DRB1) genes.

Sequencing the amplified DNA matched 99% of reference DNA samples from 40 cell lines with known HLA genotypes. In 59 clinical samples, sequencing determined HLA genotype and identified 3 new HLA alleles-2 previously undetected short insertions and 1 single base pair deletion. Genotyping of a few samples can be done within five days.

Mindrinos said the benefits of his sequencing technology are "higher resolution, lower cost and higher throughput. This technology has reduced the cost of HLA typing and improved its accuracy such that it can soon be part of a standard patient profile, equivalent to a blood type." He told SciBX that the method is less expensive because it can analyze more than 2,000 samples per instrument run, which is over 1,000 samples more than competing high throughput sequencing technology.

According to Mindrinos, applications of the technology include matching transplantations, evaluating responses in clinical trials of therapeutics and vaccines, and conducting disease association studies. His group is now working to extend the HLA genotyping method to cover the four other HLA genes.

He said Stanford University is filing a patent application. The technology is unavailable for licensing.          -LM