1. HLA-Class I: Typing Theory

2. 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

3. 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
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

4. 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

5. 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

6. 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

7. 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

8. 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

9. Example HLA-C Amplification primer with QIAGEN Long Range Polymerase2 3 4 5 6 7 8
3’UTR 1
5’UTR
Amplification primer with QIAGEN Long Range Polymerase
Core Sequencing primer 9 9

10. HLA-C: Allele Ambiguity10 10

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

12. HLA-B: Genotype Ambiguity12 12

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

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

15. 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

16. 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 ( kb)
HLA-DQB1 ( kb)
HLA-DPB1 (5.0 & 5.7 kb)
HLA-DPA1 (4.7 kb)
HLA-DQA1 ( 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.