New strategies for efficient typing of HLA class-II loci DQB1 and DRB1 by using PyrosequencingTM

Item Type:	Article
Title:	New strategies for efficient typing of HLA class-II loci DQB1 and DRB1 by using PyrosequencingTM
Creators Name:	Entz, P. and Toliat, M.R. and Hampe, J. and Valentonyte, R. and Jenisch, S. and Nuernberg, P. and Nagy, M.
Abstract:	The characterization of genetic risk factors for complex diseases located on chromosome-6 frequently requires human leucocyte antigen (HLA) genotyping of large patient cohorts. Currently available methods do not support high-throughput HLA typing beyond the major allele group level. We, thus, developed a high-throughput approach for the HLA-DQB1 and HLA-DRB1 loci that is based on Pyrosequencing™. Pyrosequencing™ offers a higher degree of automation than direct sequencing or oligotyping. Using a dispensation order optimized for the particular HLA locus, rapid group typing and fine resolution can be achieved. We implemented the method for two important HLA loci - DQB1 and DRB1. The HLA-DQB1 typing method comprises the following steps: splitting the potential alleles after a generic polymerase chain reaction (PCR) amplification into groups with a first Pyrosequencing™ reaction and resolving the split allele groups by means of five further Pyrosequencing™ reactions. The HLA-DR gene family is known to be the most polymorphic one in the HLA class-II region because of a large number of DRB1 alleles. Because of this complex nature, HLA-DRB1 typing was performed by means of a combination of sequence-specific PCR typing and Pyrosequencing™. HLA-DQB1 typing and HLA-DRB1 typing were performed successfully by using standard DNA samples with the help of known HLA genotypes and in a blind study by using the samples from the Deutscher Zell Austausch 2002 and 2003. The approach was optimized and was practically tested for genotyping in disease association studies. Our well-elaborated Pyrosequencing™-based protocols offer a new alternative to the existing HLA class-II typing methods and represent a convenient and economic solution, a unique combination of high accuracy with high-sample throughput.
Keywords:	Disease Association, HLA-DQB1, HLA-DRB1, Sequence-Based Typing, Sequence-Specific PCR, Single-Nucleotide Polymorphism
Source:	Tissue Antigens
ISSN:	0001-2815
Publisher:	Blackwell Publishing
Volume:	65
Number:	1
Page Range:	67-80
Date:	1 January 2005
Official Publication:	https://doi.org/10.1111/j.1399-0039.2005.00345.x
PubMed:	View item in PubMed

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