Pyrosequencing-based strategies for improved allele typing of human leukocyte antigen loci.
Successful transplantation of tissue during solid organ and bone marrow transplantation relies on accurate determination of the human leukocyte antigen (HLA) phenotype of the potential donor(s) and recipient. Matching donor with recipient for a kidney transplant generally means finding a six-antigen match by looking at each of two alleles at HLA-A, -B, and -DR loci. For bone marrow transplantation the HLA-C and -DQ alleles are also considered. Molecular techniques, including sequencing, are capable of precisely defining HLA alleles. Because of the large number of possible allelic combinations there are numerous ambiguities associated with heterozygous genotypes even when sequence-based typing protocols are used. Sequencing-by-synthesis methodology employed by Pyrosequencing represents an improvement when applied to HLA genotyping that allows resolution of many ambiguous allelic pairs. Out-of-phase sequencing of HLA alleles by Pyrosequencing can resolve cis/trans ambiguities that would otherwise require the sequencing of isolated cloned DNAs. Single-nucleotide polymorphism typing of HLA for the presence of specific variants is also beneficial for monitoring HLA-encoded genetic risk to autoimmune diseases, such as celiac disease, rheumatoid arthritis, and type 1 diabetes mellitus.
Ringquist
Steven
S
Division of Immunogenetics, Department of Pediatrics, Rangos Research Center, Children's Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
Styche
Alexis
A
Rudert
William A
WA
Trucco
Massimo
M
D000483:Alleles; D001483:Base Sequence; D002896:Chromosomes, Human, Pair 6 / Q000235:genetics; D004247:DNA / Q000097:blood / Q000302:isolation & purification; D011756:Diphosphates / Q000378:metabolism*; D004587:Electrophoresis, Agar Gel; D005838:Genotype; D006680:HLA Antigens / Q000145:classification / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA / Q000379:methods*; D013048:Specimen Handling
D011756:Diphosphates; D006680:HLA Antigens; D004247:DNA