Polymorphism analysis within the HLA-A locus by universal oligonucleotide array.
Human leukocyte antigen (HLA) class I genes present some of the most complex single nucleotide polymorphism (SNP) patterns in the human genome. HLA typing is therefore extremely challenging. In this article, we use the ligation detection reaction (LDR) combined with a universal array (UA) as a robust and efficient method to analyze SNPs within the HLA-A region that includes HLA-A alleles of interest for immunotherapy in tumor diseases. The LDR, combined with a UA platform, has been optimized for the detection of 27 alleles distributed within exons 2 and 3 of HLA-A. The assay involves the amplification by PCR of the HLA-A genomic region (1,900 bp), the cycled ligation reaction, followed by the capture of ligated products through hybridization onto a UA. Each slide was designed to allow the detection of up to eight samples in parallel. The PCR/LDR/UA HLA-A assay was evaluated by analyzing 62 individuals (31 homozygous and 31 heterozygous) previously typed by direct sequencing. We demonstrate that the microarray genotyping procedure described here is a robust and efficient method for unambiguous detection of HLA alleles. HLA genotyping by PCR/LDR/UA is in perfect agreement with typing obtained by direct sequencing. Our results clearly demonstrate that the combination of enzymatic processing (LDR) and a demultiplexing hybridization onto a UA is a robust tool for SNP discrimination within the highly polymorphic HLA region. We demonstrate the specificity and efficiency of such an approach, suggesting the feasibility of a PCR/LDR/UA low resolution HLA typing procedure.
Dipartimento di Scienze e Tecnologie Biomediche and CISI, Università degli Studi di Milano, Segrate, Italy.
Clarissa
C
Consolandi
Andrea
A
Frosini
Cinzia
C
Pera
Gian Battista
GB
Ferrara
Roberta
R
Bordoni
Bianca
B
Castiglioni
Ermanno
E
Rizzi
Alessandra
A
Mezzelani
Luigi Rossi
LR
Bernardi
Gianluca
G
De Bellis
Cristina
C
Battaglia
D000483:Alleles; D016000:Cluster Analysis; D004252:DNA Mutational Analysis / Q000295:instrumentation / Q000379:methods*; D005091:Exons / Q000235:genetics; D005838:Genotype; D015234:HLA-A Antigens / Q000235:genetics*; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods*; D016133:Polymerase Chain Reaction; D020641:Polymorphism, Single Nucleotide / Q000235:genetics*; D012680:Sensitivity and Specificity
D015234:HLA-A Antigens