HLA gene amplification and hybridization
analysis of polymorphism. HLA matching for
bone marrow transplantation of a patient with
HLA-deficient severe combined
immunodeficiency syndrome.
L A Baxter-Lowe, … , J T Casper, J Gorski
J Clin Invest.
1989;84(2):613-618. https://doi.org/10.1172/JCI114206.
The treatment of choice for certain immunodeficiency syndromes and hematological
disorders is bone marrow transplantation (BMT). The success of BMT is influenced by the
degree of HLA compatibility between recipient and donor. However, aberrant expression of
HLA sometimes makes it difficult, if not impossible, to determine the patient's HLA type by
standard serological and cellular techniques. We describe here the application of new
molecular biological techniques to perform high resolution HLA typing independent of HLA
expression. A patient with HLA-deficient severe combined deficiency was HLA typed using
in vitro amplification of the HLA genes and sequence-specific oligonucleotide probe
hybridization (SSOPH). Two major advances provided by this technology are:detection of
HLA polymorphism at the level of single amino acid differences; and elimination of a
requirement for HLA expression. Although the patient's lymphocytes lacked class II HLA
proteins, polymorphism associated with DR7,w53;DQw2;DRw11a (a split of DR5), w52b (a
split of DRw52);DQw7 were identified. The patient's class I expression was partially
defective, and typing was accomplished by a combination of serological (HLA-A and -C)
and SSOPH analysis (HLA-B). Complete patient haplotypes were predicted after typing of
family members [A2;B35(w6); Cw4; DRw11a(w52b);DQw7 and A2;B13(w4);
Cw6;DR7(w53); DQw2]. Potential unrelated donors were typed and a donor was selected
for BMT.
Research Article
Find the latest version:
HLA Gene
Amplification and Hybridization Analysis of
Polymorphism
HLA Matching for Bone Marrow Transplantation of
aPatient
with HLA-deficient
Severe Combined
Immunodeficiency
Syndrome
LeeAnnBaxter-Lowe,** JayB. Hunter,* JamesT.Casper,**II and Jack Gorski**
*TheBlood Center ofSoutheastern Wisconsin,Inc.,*The Medical College ofWisconsin,and
IChildren's
Hospital ofWisconsin,Milwaukee, Wisconsin 53233
Abstract
The treatment of choice for certain
immunodeficiency
syn-dromes andhematological
disorders is bone marrowtrans-plantation (BMT).
Thesuccess of BMT is influenced by thedegree of
HLAcompatibility
between recipient and donor. However,aberrant
expression of HLAsometimes
makes itdifficult,
if notimpossible,
todetermine
the patient's HLA type bystandard serological
and cellulartechniques.
Wede-scribe
heretheapplication of
newmolecular
biologicaltech-niques
toperform highresolution
HLA typingindependent
of HLAexpression.
A patient withHLA-deficient
severecom-bined
deficiency
was HLAtyped using in vitroamplification
of theHLA
genes andsequence-specific oligonucleotide
probehybridization (SSOPH).
Two majoradvances
provided by this technology are:detection of
HLApolymorphism
at thelevel
ofsingle amino acid differences;
andelimination of
a requirementfor
HLAexpression.
Although thepatient's lymphocytes
lacked
class II HLAproteins, polymorphism
associated withDR7,w53;DQw2;DRwlla
(a split of DR5), w52b (a split of
DRw52); DQw7
wereidentified.
Thepatient's class
Iexpres-sion
waspartially defective, and typing
wasaccomplished by
acombination of
serological (HLA-A
and
-C)
andSSOPH
anal-ysis
(HLA-B).
Complete patient haplotypes
werepredicted
after
typing of family members
[A2;
B35(w6); Cw4;
DRw1
la(w52b);
DQw7 and A2; B13(w4); Cw6; DR7(w53);
DQw2j.
Potential unrelated donors
weretyped
and a donor wasselected
for
BMT.Introduction
Successful
bone
marrowtransplantation (BMT)'
is
dependent
upon the
degree
of HLAmatching
betweendonor/recipient
This workwaspreviously published as an abstract in 1988 (Hum.
Immunol.25:79).
Addressreprintrequests to Dr.Baxter-Lowe, The Blood Center of
Southeastern Wisconsin, 1701 West Wisconsin Avenue, Milwaukee,
WI53233.
Receivedfor publication27December1988and in revisedform 16 March 1989.
1. Abbreviations used in this paper: BMT,bone marrowtransplant; GVHD,graft-vs.-host disease; LCL, lymphoblastoidcelllines; PCR, polymerase chain reaction;RFLP, restriction fragment length
poly-morphism; SCID, severecombined immunodeficiency; SSOPH,
se-quence-specific oligonucleotideprobehybridization.
pairs
(1). This
resultsfrom
thephysiological
role of HLA inself-restriction of cellular interactions during
an immune re-sponse(2). If polymorphic residues in
the HLA proteins aremismatched,
theimmune
system mayrecognize
the cellsbearing the mismatched
HLA asforeign.
Theconsequences
ofsuch
mismatching
include graft-versus-host
disease (GVHD),rejection of grafts,
andfailure
to reconstitute acompetent
im-munesystem (3). Theseproblems
are minimized by selectionof HLA-matched siblings
as donors.Unfortunately,
thisop-tion is available for
only
30-40% of
patients
who could
benefit from
abone marrowtransplant.
In theremaining
pa-tients
(60-70%),
HLAtyping with
high resolving power is nec-essaryfor selection of
anoptimally matched,
unrelated donor (1,3).
Traditionally,
HLAtyping has been accomplished
bysero-logical
orcellular techniques, which require the
presenceof
detectable levels
of
HLAproteins
onthe
surface of
lympho-cytes. In some cases(e.g.,
HLA-deficient
SCID
orcellular
de-pletion due
tochemotherapy), the levels
of
the
HLAproteins
ornumber
of available cells
areinadequate
toachieve
reliable
HLA
typing.
Another
limitation
of
traditional
typing
methods
is the
inability
toresolve all
functionally important
HLAal-leles. These circumstances have
prompted
the
development of
methods for analysis
of
HLApolymorphism
atthe
genetic
level (4, 5).
We
describe here the first example
of
the
application
of
tworecently developed techniques,
geneamplification
and
oligo-nucleotide
hybridization,
todetermine the
HLA typeof
aCaucasian
patient
with HLA-deficient
severecombined
im-munodeficiency (SCID).
This
disorder is characterized
by
combined
immunodeficiency associated
with
defective
ex-pression of
class
Iand/or
class
II HLAproducts
onmononu-clear cells.
Early
reportshave described
this
disorder
asbare-lymphocyte
syndrome (6).
However,
recentreportshave used
the
term"HLA-deficient
SCID"
toacknowledge
the presence
of
non-HLAproteins
onthe surface of mononuclear cells.
Without correction of the
disorder,
the
immunodeficient
pa-tient will succumb
tooverwhelming infection, usually during
the
first few
yearsof life
(6). Although
BMTis the
treatmentof
choice,
donorselection is
impeded by
the
inability
to usestan-dard
serological
methods
todetermine
the
patient's
HLA
type. Weresolved this
problem
by
instituting
newmolecular
biolog-ical
techniques for
HLAtyping.
HLA geneswereselectively
amplified (7)
from
DNAisolated from
thepatient, family
members, and
prospective
unrelated
donors.Polymorphic
res-idues
in
theamplified
DNAwereidentified
by
sequence-spe-cific
oligonucleotide
probe
hybridization (SSOPH).
SSOPH
candiscriminate
single
basepair
mismatches
(5),
which is
equivalent
todetection of
asingle
amino acid
polymorphism
in the HLAproteins.
This
caseillustrates
theutility
of SSOPH
incircumstances
inwhich
expression
of
HLAproteins
is
re-duced
and/or fine resolution of
allelesis
required.
HLATypingwithOligonucleotideProbes 613
J.Clin.Invest.
© TheAmerican
Society
for ClinicalInvestigation,
Inc.0021-9738/89/08/0613/06
$2.00
Methods
Oligonucleotides. Oligonucleotides were synthesized by a Gene As-sembler(Pharmacia Fine Chemicals, Piscataway, NJ) using phosphor-amidite chemistry. Oligonucleotides were purified using OPC
car-tridges(Applied Biosystems, Foster City, CA)orgel electrophoresis. Cell lines. Lymphoblastoid cell lines (LCL) used here were distrib-uted andcharacterized by the 10th International Histocompatibility Workshop.
Serological methods. Heparin-treated venous blood wasincubated
with carbonyl iron, and peripheral blood lymphocytes (PBL) were
obtained by centrifugation over Ficoll-Hypaque gradients (1.077 g/ml). Cells were washed, T and B cellswereseparated by nylon adher-ence, and HLA typingwasperformed by standard
microlymphocyto-toxicityassays(8, 9). Serological specificitieswereassigned according
toWHO nomenclatureadopted followingthe 10thInternational
His-tocompatibilityWorkshop(10).
DNA isolation and
amplifcation.
DNA was isolated from blood cellsasdescribedpreviously (11). Primer-directed enzymatic amplifi-cation of DNAwasaccomplished usingthepolymerasechain reactionasdescribedpreviously(7).Briefly,I
00-;d
reaction mixtures contained 1.0-3.0Mg genomic DNA; 200 MM eachdeoxynucleoside 5'-triphos-phate; 1 MMeachprimer; 50mMTris-HCI, pH 8.3; 1.5 mM MgC12; and 0.01%(wt/vol) gelatin. Primers foramplification of segments of the HLA genesaredescribed in Table I.Sampleswereboiled for 1 min,transferredto a 94°Cheatblock,and 1-2UofTaq polymerase (Per-kin-ElmerCetus, Norwalk, CT)wereadded. Thereactions consisted of 30-50 cycles of denaturation (94°C),annealingfor 1-2 min (35°C for HLA-DQ; 55°C for HLA-B and -DR), andpolymerization(72-74°C) inaprogrammable heat block (Perkin-Elmer Cetus Instruments). The total time required for the PCR reaction is 3-5 h and theoretically
results in a billion-fold amplification. The products of the reaction werecharacterized by agarose gel electrophoresis of samples (7-20 ul) followed by detection ofamplified DNAwith ethidium bromide
staining.
Hybridization ofoligonucleotides.Aliquots of the reaction mixture were denatured by incubation in 0.4 M NaOH, 0.6 M NaCl for five min; neutralized by addition of two volumes ofI MTris-HCI, pH 7.0; andappliedtoGenescreen Plus Membranes (New England Nuclear, Boston, MA) using a slot blot apparatus (Schleicher and Schuell, Keene, NH). Membranes were baked at 80°C for 15min.32P-labeling ofoligonucleotides (12), hybridization, washing of membranes, and autoradiography were performed as described previously (13), except for the following minor modifications. Filters were prehybridized in 5X Denhardt's (I X Denhardt's is 0.2 mg/ml each albumin,
polyvinyl-pyrrolidone,and Ficoll), 5X SSC(IXSSC is 0.15 M NaCl, 0.0 15 M Na
citrate,pH 7.0), 10 mM Naphosphate, and 5 mM EDTA for 1-3 h at
68°C. Filterswere hybridizedovernightin5x SSC,
lOX
Denhardt'ssolution, 20 mM Na phosphate, 100Mg/ml herring DNA, 5 mM EDTA, 7% SDS, and 0.5-1.1 X 106 cpm/ml oligonucleotide probe. Totalprocessing time from receipt of samples to development of auto-radiograms can be 2 d.
TableLPrimers for
Amplification
ofFirstDomain Exon*Name Sequence
DRBETA,(16-23) 5'-ATTTCTTCAATGGGACGGAGC
DRBETA(87-94) Y-CGCCGCTGCACTGTGAAGCTCTC DQ BETA, (14-20) Y-TGTGCTACTTCACCAACGGG DQ BETA (83-89) 5'-CGTGCGGAGCTCCAACTGGT
Class1(1-6) 5'-GCTCCCACTCCATGAGG
Class 1(85-91) 5'-CGGCCTCGCTCTGGTTG
*The names of the primers include numbers that correspond to the
locationof the amino acid residues that are encoded by each primer.
Table II.
Oligonucleotide
Probes*Reference for Probename Probesequence sequence of allele
DRLocus
E58 GCCTGATGAGGAGTACTGG 14, 15, 16
L30 CTGGAAAGACTCTTCTATA 17 D70.1 CTGGAAGACAGGCGGGCCG 14
H30 CTGGAGAGACAC 18
F37 GGAGTTCGTGCG Gorski,
unpublished
DE 70 CTGGAAGACGAGCGGGCCG 18FL37 GGAGTTCCTGCG 18
A70 CTGGAGCAGGCGCGGGCCG 12, 19 H33 TACTTCTATCACCAAGAGG 20
N 77 GGACAACTACTG 18
ErCV 28 CTGGAAAGATGCATCTATA 21
N 1126 GGAACCTGATCA 20
dsdvge 41 GACAGCGACGTGGGGGAG
DQLocus
E 45 GGAGGTGTACCG 22
EF 46 GGGGGAGTTCCG 23
iynre 31 CTATAACCGAGA
BLocus
A46 CGAGGATGGCGCCCCGGGC 24 egpeyw 55 GGAGGGGCCGGAGTATTGG
* Probe sequencesweredesignedbyidentifying allele-specificand
consensusregionsinpreviously publishedsequences forHLA-DR,
-DQ, andBloci.
This probedoes nothybridizewith thenonpolymorphic B4 locus.
Selectionand nomenclatureof oligonucleotide probes. Sequences
usedforgenerating oligonucleotideprobeswerederivedfrom
compari-son ofpublishedandunpublisheddata. Asequence conserved in all alleleswasusedtoprepareacontrolprobe. The sequences of all known alleleswerealignedandregionswerechosen that could identify indi-vidual alleles. Some alleles do not contain a unique sequence and must beidentified withacombination of probes that detect sequences
pres-entintwo or morealleles. Theoligonucleotide probes used in the case presented hereare listed in Table IIalong with the references from which the total sequence data were originally reported. The oligonucle-otide probes are namedaccordingtothecorresponding amino acid sequencesusing single letter symbols. Consensus probes are named in lower caseletters. Thus, the DR-#consensus sequence is named "dsdvge." Polymorphic probesare designated by upper case letters
symbolizingtheresidue(s)thatdiffer(s) from the consensus sequence followed byanumberindicating the position of the first polymorphic residue in the name of the probe. For example, the probe "E 58" encodes the consensus sequence with the exception of the glutamic acid (E) codon atposition 58. The polymorphic probes can be asso-ciated withone or afewHLA-DRallelesas indicated by comparison of nucleic acid sequences. When a single serological specificity is asso-ciated withmultiple nucleic acid sequences, the alleles are given the
serologicaldesignation followed by a lower case letter (e.g.,DRwlla
andDRwllb).
Results
manuscript
inpreparation). Briefly,
thediagnosis
of HLA-defi-cient SCID was based on the infant'simmunodeficiency
man-ifested by
aprogressive pneumonia
due to Pneumocystis cari-nii, absence of serum immunoglobulins and lack of expressionof
HLAantigens
on the mononuclear cells. The HLAdefi-ciency
was indicated by the lack of HLA on the cell surface (byFACS
analysis using anti-,32 microglobulin
and anti-DRmonoclonal antibodies)
andfailure
todetect class I or class IIantigens by
serological typing methods. Initially, HLA typingofthe patient samples
was completely negative except for weakreactions with
twoof six HLA-A2 antisera. After culturing
cells
with avariety
of mitogens and lymphokines,HLA-C
could be detected,
andthe
HLA-Atyping
improved.Products
ofthe HLA-B, -DR,
and-DQ loci
were not detectable after anyprocedures.
The HLA-DR, -DQ, and -B types
of
the patientwere
de-termined
by
SSOPH ofamplified
DNA. Polymorphicregions
of
the HLA genes wereamplified from genomic
DNA usingthe
primers described in Table I. Electrophoretic analysis of
the
products of each amplification
revealed asingle ethidium
bromide staining band of predicted size (data
notshown).
Amplified
DNA,from
casesamples and acontrol panel,
wasanalyzed by SSOPH with the probes listed
inTable II.
Oligonucleotide
probes
weredesigned
todetect
polymor-phisms that
areassociated with
HLAphenotypes that wouldbe
expected
inthe
patient, based
onserological analysis of
samples from family members.
The relevant HLA-DR typesdetected in
family members
wereHLA-DR2,4(w53),7(w53),
and
wII(w52).
Apreliminary SSOPH for DR2,4,7,w
11and
w52
indicated that the patient
wasDR7,wl l,w52.
The HLA-DRregion
of the major histocompatibility complex of
DR7and
DRwl 1individuals
usually contains
twoloci
encoding
DR,B
chains.
The
DR7and
DRwl 1products
areencoded by
the B1
locus, and the supertypic specificities DRw52 and
DRw53
areencoded by the B3 and B4
loci,
respectively.
There arethree
different
B1 locus
sequencesthat
cangive
rise
to anHLA-DRwl
1 serotypeand three different B3
sequencesthat
areassociated
with the DRw52
specificity.
Asecondlevel
of
SSOPH
provided
discrimination between the three
possible
types
of
HLA-DRwl 1 sequences and threepossible
DRw52
sequences.The
resulting hybridization
pattern is consistent
with the presence ofsequences derived from
celllines that weretyped
as DRwlla and DRw52b. SSOPH
wasalso
utilized toconfirm the
presenceof
anticipated
allelesof HLA-DRw53,
HLA-B(B13), and
HLA-DQ (DQw2
and
DQw7).
An
example of primary SSOPH
datais
shown in Fig. 1.The
patient
(child)
rowshows positive hybridization with
threepolymorphic
probes, D70, E58, and L30. Positive
hybridiza-tion
of
the E58
probe is consistent
with the
presenceof
a DRwl 1allele. The
specificity of hybridization
of the E58
probe is demonstrated in the experimental control
shownin
Fig. 1, bottom. E58 hybridizes to DNA from a homozygousDRwl
1 cellline but does
nothybridize
to DNAderived from
acontrol
panel of cells
expressing
other HLA-DR alleles. The presenceof
aDRwl
1 subtype in thepatient
is alsoindicated
by
hybridization
with the
D70probe. This
probehybridizes
with HLA-DRw8
andwith
the-DRw1
la
allele (see controlpanel
in
Fig.
1,bottom), thus
indicating
that thepatient is
DRwl
la. This
alleleis
alsopresent in thepatient's father
andpaternal
grandfather.
Asimilar
process can be used tointerpret
thehybridization
resultsusing
the other twoprobes. Probe L30A
1D701 A701E58
L30
Child
_
Mother
dFather
-Aunt
-M.
Grandmother
1/2
Sibling
-P. Grandmother
P.
Grandfather
-B
DR TYPE
I
SOURCE
I
D701
A70 |E58
I
L30
DR WT 100
DRI67
W21 DR15, W2DR16, W22 DR3, 520 DR4, W4
DR4, WIO DR4, W14
DRII, W5, 52b DR13,
W19,52c
DR14,
W9,
52bDR7,
W17DR4, 8 DR9, W23
WJR
AMAI RML
STEIN
WTS YAR PE 117
SWEIG EMJ
TEM
MOU
JON
DKB
4W
Figure1.SSOPHanalysisofamplifiedDNA.(Top)An autoradio-gram of SSOPHanalysisofpatient (child)andfamilymembersis shown. Eachrowshows
hybridization
resultsderivedfrom thesourceof theDNAthatis listedattheleft.TheDNAin each column has been
hybridized
with theoligonucleotideprobe indicatedatthetop of the column.(Bottom)Theautoradiogramshowshybridization
controlsusingDNAderivedfrom wellcharacterizedcells whose name,DRandDwtypearegivenatleft.Allcells except JON and
DKB arefrom thecoreLCLpanel from the TenthInternational
His-tocompatibility Workshop. Oligonucleotide probesused in
hybrid-izationsareindicatedatthe top of each column.
hybridization
withpatient
andmaternal
DNA indicates the presenceof
DR7.The A70probe indicates
the presenceof
anHLA-DR2
allele, which is detected in
the aunt,half-sibling,
maternal grandmother,
andpaternal grandmother.
Asshown inFig.
1, bottom, allhybridization experiments
includecon-trolsto
confirm
thespecificity
of
hybridization.
Asummaryof
the
hybridization
datafrom
theHLA-DR,
-DQ,
and-Bloci is
presented
inFig.
2.Combining
thepatient
andfamily
data,
the class IIhaplotypes
of
thepatient
werededuced
asDRwl
la(w52b);DQw7/DR7(w53);DQw2.
Thesehaplotypes
C M F mGM mGF pGM pGF 1/2S - + -+ _ - + _ + _-_ + _-+ _ _ + -- +- -_ _ +
+ - + + 4.
-+ - -+ + + - + + + + + + + +-+ + A +
- + n - + + n n
+ - n + - + n n
+ + n + + + n n
DI D2
+ +
+
-+ +
+ +
+ - 141 +
+ + + + + + + + +I +
DR5(wl1) DR7
DR5(wi 1),DR8
DRwSb DR7, DRw52c, DR6Dw9 DR6(w13); DR4Dw10; DRwl1b DRW52B
DR1 DR2(w15, w16) DF13 DR4 DRw53 DRbeta DOw7 DQw2 DObeta B13 Aand B
Figure 2.Summary ofSSOPH data from
patient,
family members,
and unrelated donors.Thesourceof thesamplesislistedacrossthetop(C-patient; M-mother,
F-father;
mGM-maternalgrandmother;
mGF-maternalgrandfather; pGM-paternal grandmother;pGF-pater-nalgrandfather; 1/2S-halfsibling;A-aunt, andDl andD2-unrelated donors). Theprobesfor each locusareindicatedattheleft. The
asso-ciationofeachprobewith known HLA allelesisshownattheright.
Boxesindicate the best match betweenpatientandpotentialdonors. With the probes used,potentialunrelated donors showedan identi-calpolymorphism
profile
forHLA-DR,butonlydonor1(Dl)
wasidentical for all three loci.
are consistent with established
linkage
disequilibrium
for thecaucasian population.
Samples
from the patient's
family
members
weretyped
byboth
serological
and SSOPH methods. The
oligonucleotide
Figure3.Haplotypes of patientandfamily membersdetermined by
serologicalandSSOPH analysis. Serological specificities(topleft)and SSOPH(top right)areindicated foreachhaplotype derived fromthe
pedigreeofpatient's family (bottom).
hybridization
data correlated
perfectly
with the HLA types identifiedserologically (Figs.
2 and3).
Inaddition,
the
sero-logical typing
of thepatient's family
andanalysis of
thesegre-gation
ofHLA alloweddetermination
of thehaplotypes
ofthe
patient's family (Fig. 3).
None
of thefamily
members
wereHLA-identicaltothe
patient (Fig. 3).
Asaresult,
asearch for
awell-matched
unrelated donor wasinitiated. The search
re-vealed
twopotential donors who
wereHLA-typed by both
serological
and SSOPH methods. Thisinformation
was uti-lizedtoselectthe bestpossible
donor fortransplantation. The
serological typing
of theselected donor
wasA2, 11;B1
3,35,
(w4,6);DR7,wl 1,(w52,w53);DQw2,w7, and the
SSOPH pat-ternwasassociated withB13;DR7,wl la,w52b,w53;DQw2,w7
(Fig. 2).
Thepatient
wastransplanted and engrafted readily
asdetermined by chromosomal analysis,
normalclass
I and II HLAexpression,
anddetection of
HLA-Al 1. Thepatient is
presently alive, 39
wkafter transplantation
andis
infection
free.
She isdeveloping
normally with
resolving
mild/moderate
(skin, liver) graft-vs.-host
disease.
Discussion
The use of DNA
amplification
andSSOPH
toaccomplish
HLA
matching
fortransplantation
isdemonstrated
in thisstudy. Although
limiteduse ofthesetechniques
has beenre-ported
for purposes of disease association and forensicidenti-fication,
this is the firstexample
ofcomprehensive
HLAtyping
and
subsequent
useof thetyping
forHLAmatching
inBMT. This new approach for HLA matching providesenhancements
that will have greatutility
in selection of donors for BMT. One important advantage provided by this approach islatitude
inquality, quantity,
andsourceoftissue foranalysis.
This will be valuable in numerous cases in which it is notpossible
toobtain
asufficient
numberof normallymphocytes
(e.g., as a result of HLAdeficiency
orchemotherapy)
to achieve reliable HLAtyping by
conventional methods. A second majoradvantage
of SSOPHmethodology
is explicit definition of alleles. At thistime,
serological reagents cannot provide discrimination be-tweenall known HLA alleles. In contrast, SSOPH candetect
allpolymorphism
atthe level of single amino acidresidues.
Suchmicropolymorphism
may influence the outcomeof
transplantation;
thus matching at this level may be very bene-ficial.The case presented here
demonstrates
theusefulness of
DNA amplification and SSOPH inmatching
for BMT. In an HLA-deficient SCID patient,serological
HLA typing of freshly prepared cellsfailed
to detect most HLAproducts.
Mitogen/
lymphokine
treatment was unable toinduce
detectable levels ofHLA-B,
-DR, or -DQ products.However, analysis
of the patient's DNA by SSOPH, which is notdependent
upon pro-tein expression, waspossible.
The combinedutilization
ofse-rological
andSSOPH
analysis allowed detection of HLA-A2; B 13;Cw4,w6; DR7,w
11a,w52b,w53;DQw2,w7.
B35 was not testedfor,
but ispredicted
by thehaplotypes
of family members. It was notpossible
to test for the presence of the B35 allele because the sequence of this allele has not yet been determined.SSOPH
identifies relevant
HLApolymorphism
and detects single basepair
differences thatcorrespond
tosingle
aminoPROBE DRLouss E58 L30 D70.1 H30 F37 DE70 FL38 ErCV28 A70 N77 H33 Nil26 dmdvge4l QQLocus E45 EF46 ye31 B Locuss A46 egpeYw55 + + + + + + + +
Serology
SSOPH
go ,PatDQDO a A2;B36(w6);Cw4;DRw1 1(w52);D0w7 E 58; DE 70; H 30 E45 b A25;841(w6);Cw-,DR4(w53);DQw7 H33,NIl26 E45
c A2;35(w6);Cw4;DRw15;DQwI A70
d A2;B13(w4);Cw6,DR7(w53);DQw2 L30; F37, NI26 EF46 A46 e A31;B60;Cw3;DR3(w52);DQw2 FL3 7; N 77 EF46
f A24134(w4);Cw-,DRw15;DQwl A70 g A2;B7(wG;Cw7;DRw15;DQw1 A70 h A2;B4 w4);Cw-;DRw13(w52);DQw1 FL37; DE 70
acid
differences in theproteins.
Thisisamarkedimprovement
overanother method of DNA
analysis
that detectsrestrictionfragment
length polymorphism (RFLP). Most of the
RFLPresults
from polymorphism
innoncoding
sequencesof
the ge-nome.Therefore,
RFLP does notdirectly
measurefunctional
polymorphism,
but
relies
uponlinkage
disequilibrium
with
the
coding region (4).
RFLPanalysis
has been
usedfortyping
aSCID
patient (25),
but it is less amenable to routinetyping
because
it is labor intensive.Furthermore,
RFLP data fromheterozygous
individualsaredifficulttointerpret
(26).
An
important
asset of SSOPHmethodology
is thehigh
level of resolution
of HLA alleles obtained. In the casepre-sented
here,
SSOPH
wasable
todistinguish
between threepossible
types of DRw 11 alleles(14,
15)
and threepossible
types
of DRw52 alleles
(18, Gorski, J., unpublished
observa-tions).
This level of resolution was useful in donor selectionbecause
itconfirmed that both donor andrecipient
shared the samesubtypes, DRwl
la and DRw52b.Matching
with ahigher degree of
specificity
should
result in
lessgraft
rejection
and
reduction in
frequency
and
severity
of GVHD.
Definition
of
HLApolymorphism
atthe level of
single
amino acid
differencesprovides
the basis for afunctionally
relevant
typing
system.By
retrospective
and
ongoing
SSOPH
analysis
of
BMTdonor/recipient
pairs,
eachpolymorphism
can be
evaluated
for its effect onthe outcome oftransplanta-tion.
Inaddition,
each
polymorphism
canbe correlated
withthe
extentof
serological
and cellularalloreactivity.
Finally,
each
polymorphism
can bemapped
ontothe
recently
eluci-dated
HLA structure(27,
28).
This information canbecom-bined
togenerateascale
rating
eachposition
according
toitsfunctional
importance.
Allowable mismatches may beidenti-fied
using
this
system,and
use of this information indonor
selection
could
ultimately
increase the
numberof
successful
unrelated donor
transplants.
Acknowledaments
Wethank the members ofourlaboratories for their excellent technical
contributions, particularlyA.Bissonette,P.Fischer, S.Hackbarth,K.
Huston,P.Kirchner,G.Knorr,andD.Schmidt. We also thankDr. P.
Newman for histhoughtfuldiscussions
regarding
DNAamplification,
Dr.R.Ashfor hisencouragementof research relevanttobonemarrowtransplantation
and Drs. R. Asterand D. Eckels for their support of SSOPH analysis ofHLApolymorphism.
We also acknowledge M.Iversonand J.Trubyfor their assistance in
preparation
ofthemanu-script.
Supported by The BloodCenter ofSoutheastern
Wisconsin,
Na-tional Institutes of Health(AI-26085-01)
and the Midwest AthletesAgainstChildhood Cancer Fund(MACC).
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