HLA-Class I: Typing Theory

SBTexcellerator® Accelerating High Resolution HLA Typing by Excellent Performance Single PCR Easily solve ambiguities Add. exon seq. primers Group Specific Seq. Primers Primers and Protocol 2

Extracellular domain a3 q-arm p-arm chromosome 6 HLA region class II class II class III class I class I DP DQ DR B C A B C A 1 2 3 4 5 6 1 2 3 4 5 6 7 8 leader peptide 5' UTR leader peptide 5' UTR extracellular extracellular domain b 1: domain a 1 and peptide a 2: peptide binding groove binding groove extracellular Extracellular domain a3 domain b 2 transmembrane Transmembrane region region cytoplasmic tail cytoplasmic tail class II class I

Sequencing Based Typing High-resolution method Uses all nucleotides to determine HLA alleles No changes in strategy needed with IMGT/HLA update Detection of new alleles Limited number of reactions 4

SBT Strategies Core typing: sequencing essential regions HLA-A, B: Exons 2,3 and 4 HLA-C: Exons 2 and 3 DRB: Exon 2, DRB4 also exon 3 DQB1: Exons 2 and 3 DPB1: Exon 2 5 5

HLA Nomenclature HLA-A*02:01:01:01 HLA-A2 Serology HLA-A*02 Allele group main specificity/ serological equivalent HLA-A*02:01 Allelic subtypes: differences in 3rd and 4th digit = different protein HLA-A*02:01:01 Non-coding variation Differences in 5th and 6th digit = different nucleotides, identical protein Silent variation HLA-A*02:01:01:01 Variation outside coding regions Differences in 7th and 8th digit = variation outside coding regions Intron variation, splice sites, promotor 6 6

HLA Nomenclature Additional characters: N Null alleles HLA-A*24:09N No expression Premature stop codon Splice sites L Low expression: HLA-A*24:02:01:02L S Secreted: HLA-B*44:02:01:02S Q Questionable expression: HLA-A*32:11Q Unconfirmed aberrant expression 7 7

HLA Class I Strategy 2 3 4 5 6 7 8 3’UTR 1 5’UTR Amplification primer with QIAGEN Long Range Polymerase Core Sequencing primer Extended Region Sequencing primer Group Specific Sequencing Primer 8

Example HLA-C Amplification primer with QIAGEN Long Range Polymerase 2 3 4 5 6 7 8 3’UTR 1 5’UTR Amplification primer with QIAGEN Long Range Polymerase Core Sequencing primer 9 9

HLA-C: Allele Ambiguity 10 10

HLA-C: Allele Ambiguity All ambiguities resolved ! 11 11

HLA-B: Genotype Ambiguity 12 12

HLA-B: Genotype Ambiguity GSSP B20 + B26 + B16 All ambiguities resolved ! 13 13

Reagent Box Set-up 2 3 4 5 6 7 8 3’UTR 1 5’UTR EXTENDED CORE FULL 14

SBTexcellerator® Single PCR reaction per HLA locus For HLA Class I coverage of the complete gene For HLA class II coverage of exon 2 (and exon3) Primers provided for amplification and sequencing Core regions Extended regions to resolve allele ambiguities GSSP’s to resolve genotype ambiguities 15

Amplification strategy HLA-A (3.1 kb) 1 8 7 6 5 4 3 2 UTR HLA-B (3.4 kb) HLA-C (3.4 kb) HLA-DRB1 (3.7-4.8 kb) HLA-DQB1 (3.7-4.1 kb) HLA-DPB1 (5.0 & 5.7 kb) HLA-DPA1 (4.7 kb) HLA-DQA1 (5.4-5.8 kb) HLA-DRB3 (3.8 kb) HLA-DRB4 (0.4 & 1.3 kb) HLA-DRB5 (4.0 kb) SBTexcellerator amplification primer Amplified exon Exon not amplified Class I amplified entirely Class II large introns, not all exons amplified.