POSTGRADUATE
SCHOOL OF MEDICINE
HLA TYPING
D Middleton
MDSC175: Transplantation Science for Transplant
Clinicians (Online)
A MEMBER OF THE RUSSELL GROUP
CONTINUING PROFESSIONAL DEVELOPMENT
HLA TYPING
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Genes and Chromosomes
•
The MHC is a cluster of genes located on the short arm of
chromosome 6.
•
Class I genes (HLA-A,-B & -Cw) encode antigens which present
peptide to CD8+ T-cells.
•
Class II genes (HLA-DR,-DQ & -DP) encode antigens which
present peptide to CD4+ T-cells.
•
Many Class III genes (e.g TNF-alpha, C2 & C4 complement
genes) are involved in aspects of the immune response.
HLA TYPING
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4
HLA TYPING
Major Histocompatibility Complex
Chromosome 6
Tel
Long arm
Cen
Short arm
Tel
HLA Region
6p21.1-21.3
Class II
DP
DM
DQ
Class III
DR
Bf
C4 C2Hsp70TNF
Class I
B C
E
Gene map of the human leukocyte antigen (HLA) region
Expert Reviews in Molecular Medicine © 2003 Cambridge University Press
A GF
HLA TYPING
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HLA Polymorphisms
•
Most polymorphic system in the genome
•
Related to function of peptide presentation
•
Most polymorphism in peptide binding region driven by
natural selection
•
High level of coding (non-synonymous) mutations in PBR
•
Some alleles common (gene freq >0.1%), others rare
•
Ethnic variations
HLA TYPING
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HLA Class I Gene (A,B,C)
Peptide binding cleft
molecular domains
5’UT SS
1
α2
α1
α3
β2 - microglobulin
1
2
3
TM
2
3
4
5
polymorphism in exons 2 & 3
encoding 1 and 2 domains
C1 C2 C3
6
7
8
HLA TYPING
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HLA Class II B Gene
(of the Class II A genes, only DPA and DQA are significantly polymorphic)
Peptide binding cleft
β1
α1
β2
α2
molecular domains
5’UT SS
1
1
2
2
3
polymorphism in exon 2
encoding 1 domain
TM/C
4
C
5
3’UT
HLA TYPING
Linkage Disequilibrium
The observation that
two or more alleles
at two are more loci
in a population are
associated more or
less frequently than
would be predicted
from their individual
frequencies.
The non-random
association of MHC
alleles in a
population.
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HLA TYPING
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Structure HLA Class I
Peptide binding cleft
α2
α1
α3
β2 - microglobulin
 polypeptide chain with 3 domains + 2 microglobulin
HLA TYPING
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Structure Class II
Peptide binding cleft
β1
α1
β2
α2
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HLA Nomenclature
Hyphen used to separate
gene name from HLA prefix
Separator
Field Separators
Suffix used to denote
changes in expression
HLA-A*02: 101: 01 : 02 N
HLA Prefix
Gene
Field 2; specific HLA
protein
Field 1; allele group
Field 4; used to
show differences in
a non-coding region
Field 3; used to show a synonymous DNA
Substitution within the coding region
© SGE Marsh 04/10
HLA TYPING
Why HLA Type?
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HLA TYPING
HLA A+B+DR Mismatches
First Cadaver Kidney Transplants 1985-2000
Collaborative Transplant Study
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HLA TYPING
HLA A+B+DR Mismatches
Deceased Donor Kidney Transplants 2005-2010
Collaborative Transplant Study
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HLA TYPING
DNA Typing HLA-DRB Mismatches
Cadaver Kidney Transplants 1985-2000
0 Mismatch HLA-DR Serology
Collaborative Transplant Study
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HLA TYPING
HLA A+B+DR Mismatches
First Orthotopic Heart Transplants 1985-2000
Collaborative Transplant Study
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HLA TYPING
HLA-A+B+DR Mismatches
First Liver Transplants 1988-2000
Collaborative Transplant Study
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HLA TYPING
HLA A+B+DR Mismatches
First Cadaver Kidney Transplants 1985-2000
Cold Ischemia ≤6 Hours
Collaborative Transplant Study
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HLA TYPING
Cold Ischemia Time – Shared/Local
First Cadaver Kidney Transplants 1985-2000
0 and 6 HLA-A+B+DR Mismatches
Collaborative Transplant Study
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HLA TYPING
Inheritance Chart
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HLA TYPING
Kidney Transplants
First Grafts 1995-2004
Collaborative Transplant Study
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HLA TYPING
Unrelated Living Donors 1995-2005
HLA-A+B+DR Mismatches
Europe or North America
Collaborative Transplant Study
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HLA TYPING
HLA-A+B+DR Mismatches
Deceased Donor Kidney Transplants 1985-2005
Collaborative Transplant Study
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HLA TYPING
Donor Age
Related Donor Kidneys, First Grafts 1995-2005
Collaborative Transplant Study
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HLA TYPING
Donor Age
Deceased Donors, First Grafts 1995-2005
Collaborative Transplant Study
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HLA TYPING
HLA-A+B+DR Mismatches
Deceased Donor Kidneys Transplants 1985-2005
Donor Age <40
Collaborative Transplant Study
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HLA TYPING
How to HLA Type
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HLA TYPING
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SSP
5’
3’
5’
3’
3’
5’
Primer match
Primer mis-match
Amplification
Specific
products
Positive
control
Agarose gel visualisation
3’
5’
No
Amplification
HLA TYPING
PCR-SSP Phenotype
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HLA Typing by PCR-SSOP
Single generic PCR (using conserved sequence for
primers)
Hybridisation with multiple oligonucleotide probes on
solid support (tray/membrane/bead)
Positive/negative hybridisation identifies
presence/absence of allele or allele group
HLA TYPING
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Principle of PCR-SSOP
(using HLA-A as an example)
EXON 2
intron 2
EXON 3
A*01
A*02
A*03
A*11
A*24
allele-specific probe sequences
conserved sequence ( = 1 generic PCR per locus)
HLA TYPING
HLA Typing by PCR-SSOP
1. patient DNA locus specific PCR
(e.g. one primer is biotin labelled)
2. single stranded PCR product
(NaOH or asymmetric PCR)
3. hybridise with specific
probes bound to tray/membrane/
Luminex beads
4. detection of bound DNA
(e.g. streptavidin)
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HLA TYPING
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PCR-SSOP by Luminex
•
Polystyrene beads of uniform size are used as the solid phase
•
Each bead is dyed with two different fluorochromes – ratio
•
Gives 100 distinctively coloured bead populations
•
SSO’s attached to Luminex beads, multiplex reactions
HLA TYPING
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HLA TYPING
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2nd Generation Sequencing
•
High seq capacity allowing parallel analysis of amplicons for all
relevant exons
•
250bp read spans most of relevant regions
•
Pooling of amplicons from different individuals (48) needed to
make cost effective
•
Software needed for phasing of the amplicons and to filter out
related sequence reads- coamplified
•
Tissue Antigens 74,393-403
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Which Method to Use
Clinical Urgency
Combination of
methods
Staff skills
Equipment
availability
Clinical
Resolution
Sample numbers
Budget
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HLA TYPING
Matching 1
•
Difference in AA. What is important?
•
Quantity
•
Position = Function
•
Which Loci
•
Resolution required
•
By epitopes not by current nomenclature
•
Rationalisation of alleles we test for
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HLA TYPING
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Matching 2
•
Different approach for different patients
•
Difference immunogenicity of HLA mismatches
•
Genetic factors influencing immune response? (including
innate response and immunosuppressive sensitivity)
•
Intelligent mismatching
•
Acceptable mismatch program
HLA TYPING
Number of HLA Antigens and Alleles
1968-2008
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HLA TYPING
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www.allelefrequencies.net
Phenotype
Frequency
(%)
Allele
Frequency
(3 decimals)
Sample
Size
0.130
51
Allele
Population
A*0201
American Samoa
A*0201
Argentina Gran Chaco Eastern Toba
46.4
0.304
135
A*0201
Argentina Gran Chaco Mataco Wichi
40.9
0.216
49
A*0201
Argentina Gran Chaco Western Toba Pilaga
60.0
0.400
19
A*0201
Argentina Toba Rosario
34.9
0.192
86
A*0201
Australia New South Wales
0.261
134
A*0201
Australian Aborigine Cape York Peninsula
0.175
103
A*0201
Australian Aborigine Groote Eylandt
0.107
75
A*0201
Australian Aborigine Kimberly
0.111
41
HLA TYPING
www.allelefrequencies.net
Allele: A*0201 Frequency Distribution
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HLA TYPING
HLA Alleles In N. Ireland Population
(n=1000)
1994
2009
HLA-A
30/90
33.3%
30/733
4.1%
HLA-B
50/254
19.7%
50/1115
4.5%
HLA-C
23/78
29.5%
23/392
5.9%
HLA-DRB1
33/216
15.2%
33/608
5.4%
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HLA TYPING
Summary of Results
Locus Number
All 3135
Very Rare (%)
Rare (%)
Frequent (%)
0
1, 2, 3 (inc. seq.
confirmation)
>3
39.8
25.2
34.9
A
733
44.3
23.1
32.6
B
1115
39.3
25.7
35.1
C
391
43.7
24.3
32.0
DRB1
608
34.9
26.8
38.3
DQB1
95
44.2
26.3
29.5
DQA1
34
20.6
32.4
47.1
DPA1
27
48.1
22.2
29.6
DPB1
132
31.1
27.3
41.7
HLA TYPING
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NMDP Analysis
• Single MM (low or high res) at HLA-A,B,C,DRB1
associated with higher mortality
• Single MM at HLA-B and C better tolerated
• MM at 2 or more loci compounded the problem
• MM at DQ or DP not associated with survival
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HLA TYPING
Probability of Overall Survival by HLA
Matching for Early Disease Stage
1.0
0.9
0.8
8/8 HLA Matched (n=835)
7/8 HLA Matched (n=379)
0.7
0.6
Survival 0.5
50%
0.4
39%
0.3
28%
6/8 HLA Matched (n=241)
0.2
0.1
Log-rank p-value = < 0.0001
0.0
0
12
24
36
Months after transplant
Curtesy of Stephanie Lee (2008 BMT Tandem Meetings)
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FACULTY OF HEALTH & LIFE SCIENCES – CPD
Institute for Learning & Teaching
Faculty of Health & Life Sciences
Room 2.16A, 4th Floor
Thompson Yates Building
Brownlow Hill
Liverpool
L69 3GB
www.liv.ac.uk/learning-and-teaching/cpd
A MEMBER OF THE RUSSELL GROUP