Transcript HLA typing NEW TEMPLATE

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
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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.
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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
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HLA Polymorphisms
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Most polymorphic system in the genome
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Related to function of peptide presentation
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Most polymorphism in peptide binding region driven by
natural selection
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High level of coding (non-synonymous) mutations in PBR
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Some alleles common (gene freq >0.1%), others rare
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Ethnic variations
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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
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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
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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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Structure HLA Class I
Peptide binding cleft
α2
α1
α3
β2 - microglobulin
 polypeptide chain with 3 domains + 2 microglobulin
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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
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Why HLA Type?
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HLA A+B+DR Mismatches
First Cadaver Kidney Transplants 1985-2000
Collaborative Transplant Study
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HLA A+B+DR Mismatches
Deceased Donor Kidney Transplants 2005-2010
Collaborative Transplant Study
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DNA Typing HLA-DRB Mismatches
Cadaver Kidney Transplants 1985-2000
0 Mismatch HLA-DR Serology
Collaborative Transplant Study
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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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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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Inheritance Chart
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Kidney Transplants
First Grafts 1995-2004
Collaborative Transplant Study
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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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Donor Age
Deceased Donors, First Grafts 1995-2005
Collaborative Transplant Study
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HLA-A+B+DR Mismatches
Deceased Donor Kidneys Transplants 1985-2005
Donor Age <40
Collaborative Transplant Study
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How to HLA Type
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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
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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
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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)
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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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PCR-SSOP by Luminex
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Polystyrene beads of uniform size are used as the solid phase
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Each bead is dyed with two different fluorochromes – ratio
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Gives 100 distinctively coloured bead populations
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SSO’s attached to Luminex beads, multiplex reactions
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2nd Generation Sequencing
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High seq capacity allowing parallel analysis of amplicons for all
relevant exons
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250bp read spans most of relevant regions
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Pooling of amplicons from different individuals (48) needed to
make cost effective
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Software needed for phasing of the amplicons and to filter out
related sequence reads- coamplified
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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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Matching 1
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Difference in AA. What is important?
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Quantity
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Position = Function
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Which Loci
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Resolution required
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By epitopes not by current nomenclature
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Rationalisation of alleles we test for
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Matching 2
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Different approach for different patients
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Difference immunogenicity of HLA mismatches
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Genetic factors influencing immune response? (including
innate response and immunosuppressive sensitivity)
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Intelligent mismatching
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Acceptable mismatch program
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Number of HLA Antigens and Alleles
1968-2008
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www.allelefrequencies.net
Phenotype
Frequency
(%)
Allele
Frequency
(3 decimals)
Sample
Size
0.130
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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
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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
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www.allelefrequencies.net
Allele: A*0201 Frequency Distribution
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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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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
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20.6
32.4
47.1
DPA1
27
48.1
22.2
29.6
DPB1
132
31.1
27.3
41.7
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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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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
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Months after transplant
Curtesy of Stephanie Lee (2008 BMT Tandem Meetings)
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