Transcript Molecular Oncology

Chapter 15
DNA-Based Tissue Typing
Objectives
 Describe the structure of the MHC locus
that encodes the HLA antigens.
 Explain the role of these antigens in tissue
engraftment and rejection.
 Describe the laboratory methods used to
identify HLA antigens by serology testing.
 Describe the DNA-based testing methods
that are used for the identification of HLA
antigens.
The Major Histocompatability
Complex (MHC)
 The MHC is located on chromosome 6.
1 Mb
2 Mb
baba ba bbbba
HLA-
DP
DQ
DR
Class II
3 Mb
TNF
a b
4 Mb
B C
Class III
A
Class I
 The MHC contains the human leukocyte antigen
(HLA) and other genes.
Genes of the Major
Histocompatibility Locus
MHC region
Gene products
Tissue location
Function
HLA-A, HLA-B, HLA-C
All nucleated cells
Identification and
destruction of abnormal
or infected cells by
cytotoxic T cells
Class II
HLA-D
B lymphocytes,
monocytes,
macrophages, dendritic
cells, activated T cells,
activated endothelial
cells, skin (Langerhans
cells)
Identification of foreign
antigen by helper T cells
Class III
Complement C2, C4, B
Plasma proteins
Defense against
extracellular pathogens
Cytokine
genes
TNFa, TNFb
Plasma proteins
Cell growth and
differentiation
Class I
The Human Leukocyte Antigens
(HLA)
Human leukocyte antigens, the MHC gene products,
are membrane proteins that are responsible for
rejection of transplanted organs and tissues.
a 1 b1
HLA-D
a 2 a1
a 2 b2
a3
Cell membrane
a chain
b chain
a chain
b 2 microglobulin
The Human Leukocyte Antigens
(HLA)
 HLA-gene sequences differ from one individual to
another.
a.CGG GCC GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GGT GAG AGC TTC ACA
CGG GCC GCC GTG GAC ACC TAT TGC AGA CAC AAC TAC GGG GCT GTG GAG AGC TTC ACA
CGG GCC GCC GTG GAC ACC TAT TGC AGA CAC AAC TAC GGG GCT GTG GNN NNN NNN NNN
 Also written as:
b.
CGG GCC GCG GTG GAC ACC TAC TGC AGA CAC AAC TAC GGG GTT GGT GAG AGC TTC ACA
--- --- --- --- --- --- --T --- --- --- --- --- --- -C - -TG --- --- --- ----- --- --C --- --- --- --T --- --- --- --- --- --- -C- -TG -**
 Each sequence is a different allele.
***
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HLA Allele Nomenclature
 A standard nomenclature has been established by the
World Health Organization (WHO) Nomenclature
Committee.
Subregion
Gene region
HLA-DRB1
Gene locus
a- or b-chain polypeptide
 A small “w” is included in HLA-C, HLAB-4, and HLAB6 allele nomenclature: HLA-Cw, HLABw-4, HLABw-6.
HLA Allele Nomenclature
 HLA-typing at the DNA level requires nomenclature
for specific DNA sequences.
Gene region
Subregion
Allele family 25
HLA-DRB1*2503
Gene locus
Third allele
a-or b-chain polypeptide
 There are over 900 HLA alleles identified so far in all
loci.
HLA Alleles are Inherited in
Blocks as Haplotypes
A24
A30
A1
A6
alleles
haplotype
Cw1
B14
Cw3
B7
DR14
DR15
X
Cw1
B12
Cw7
B44
DR5
DR14
A24
A6
A1
A30
A6
A30
A24
A1
Cw1
B14
Cw7
B44
Cw1
B12
Cw3
B7
Cw7
B44
Cw3
B7
Cw1
B14
Cw1
B12
DR14
DR14
DR5
DR15
DR14
DR15
DR14
DR5
HLA-Typing
 Every person (except identical twins) has
different sets of HLA alleles.
 Transplanted organs are allografts, in which the
donor organ and the recipient are genetically
different.
 Compatibility (matching) of the HLA of the donor
and the recipient increases the chance for a
successful engraftment.
 Matching is determined by comparing alleles.
 Resolution is the level of detail with which an
allele is determined.
Serological Typing
Lymphocytes are HLA-typed by crossmatching to
panel reactive antibodies (PRA) using the
complement-dependent cytotoxicity (CDC) test.
Complement
antibody
Positive reaction to antibody
kills cells. Dead cells pick up dye.
Buffy coat
from patient
Negative reaction to antibody:
cells survive and exclude dye.
Serological Typing
Recipient antihuman antibodies are assessed by
crossmatching to donor lymphocytes.
Lymphocytes from organ
donor or lymphocytes of
known HLA types
Recipient serum
Positive reaction to antibody
kills cells. Dead cells pick up dye.
Negative reaction to antibody:
cells survive and exclude dye.
Serological Typing Using Bead
Arrays
Recipient antihuman antibodies are assessed by
crossmatching to known lymphocyte antigens
conjugated to microparticles. Results are assessed
by flow cytometry.
Beads
conjugated to
different
lymphocyte
antigens
Serum
antibodies
Positive for antibody
(Wash)
Fluorescent
reporter
antibodies
Negative for antibody
Other Serological Typing
Methods
 Cytotoxic and noncytotoxic methods with flow
cytometry detection.
 Enzyme-linked immunosorbent assay (ELISA)
with solubilized HLA antigens.
 Mixed lymphocyte culture measuring growth of
lymphocytes activated by cross-reactivity.
 Measure of HLA-protein mobility differences in
one-dimensional gel isoelectric focusing or twodimensional gel electrophoresis.
DNA-Based Typing
Methods
 DNA typing focuses on the most
polymorphic loci in the MHC, HLA-B, and
HLA-DRB.
 Whole-blood patient specimens collected
in anticoagulant are used for DNA typing.
 Cell lines of known HLA type are used for
reference samples.
DNA-Based Typing Methods:
SSOP
Sequence-specific oligonucleotide probe
hybridization (SSOP, SSOPH)
Specimen 1 (Type A*0203)
Specimen 2 Type A*0501
TAG CGAT
ATC GCTA
TAG A GAT
ATC TCTA
Amplify, denature, and
spot onto membranes
Specimen 1
Specim
en 2
Probe with allele-specific
...TAGCGAT..(A*02)
probes
Specimen 1 Specimen 2
...TAGAGAT…(A*05)
Specimen 1 Specimen 2
DNA-Based Typing
Methods: SSP-PCR
Sequence-specific PCR is performed with
allele-specific primers.
Amplification
SSP= Sequence-specific primer
controls
Allele-specific
product
SSP matches allele
Amplification
SSP
No
amplification
SSP
SSP does not match allele
DNA-Based Typing Methods:
SSP-PCR
Primers recognizing different alleles are
supplied in a 96-well plate format.
Reagent blank
Amplification control
Allele-specific product
Agarose gel
DNA-Based Typing Methods:
Sequence-based Typing
 Sequence-based typing (SBT) is high
resolution.
 Polymorphic regions are amplified by
PCR and then sequenced.
Reverse PCR primer
Forward PCR primer
Exon 2
Exon 3
HLA-B
Sequencing primers
Sequence-based Typing
Isolate DNA
PCR
clean amplicons
sequence
amplicon
Sequences are
compared to
reference sequences
for previously
assigned alleles.
Typing Discrepancies
 DNA sequence changes do not always affect epitopes.
 Serology does not recognize every allele detectable by
DNA.
 New antigens recognized by serology may be assigned
to a previously identified parent allele by SBT.
 Serology antibodies may be cross-reactive for multiple
alleles.
 Due to new allele discovery, retyping results may differ
from typing performed before the new allele was known.
Resolution Levels of HLA Typing
Methods
Low resolution
methods
Intermediate
resolution methods
High resolution
methods
CDC (Serology)
PCR-SSP
PCR-SSP
PCR-SSP
PCR-SSOP
PCR-SSOP
PCR-SSOP
PCR-RFLP
SSP-PCR + PCRRFLP
SSOP-PCR + SSPPCR
SBT
Combining Typing Results
 SSP-PCR followed by PCR RFLP.
 SSOP followed by SSP-PCR.
 SBT results clarified by serology.
Summary
 The MHC is a polymorphic locus encoding the HLA
genes.
 Antigens encoded by the HLA genes are responsible for
allograft tissue and organ rejection. Identifying and
matching alleles increases the chance of successful
organ and tissue transplant.
 HLA antigens and their corresponding sequence alleles
are determined by serological- and DNA- based
methods.
 Serology identifies functional antigen recognition, while
sequence analysis identifies genetic alleles with high
resolution.