Note: Descriptions are shown in the official language in which they were submitted.
133320~
METHOD FOR HLA DP TYPING
This invention relates to a method and co,~o~ilions for ~e~ g the HLA DP
genotype of an individual. In a preferred embodiment, the invention relates to using gene
amplification methodology disclosed and claimed in U.S. Patent Nos. 4,683,195 and
4,683,202 and the dot-blot and allele-specific oligonucleotide probe technology as
disclosed and cl~imçd in U.S. Patent 4,683,194. The mçthods and probes of the invention
specifically relate to the detection of the polyrnorphic class II HLA DP genes. The
invention relates to the fields of molecular biology, diagnostic m.oAicine, and forensics.
The class Il loci of the human major histocompatibility complex encode the HLA Dcell surface glycopr~leil1s which are e~yl~ssed on B lymphocytes, activated T
lymphocytes, macrophages, and dendritic cells. These proteins, which are individually
designated DR, DQ, and DP, are composed of an alpha and a highly polymorphic beta
subunit and are responsible for the ~I,sentalion of antigen to T cells. The variability in the
highly polymorphic beta subunit is locali7ed to the amino-t. .rmin~l extra~ ell~ r dom~in,
l 5 which is thought to interact with the T cell receptor and antigen peptide fra~nçnt~ The
genes encoding the class II HLA proteins are located on the short arm of chromosome six in
nS The genes enco~ling the HLA DPalpha and DPbeta chains are clustered at the
centromeric end of this region and, perhaps due to low levels of expression, were the last
of the HLA D genes to be discovered and are, therefore, the least well characterized. The
structuresj sequences, and polymorphisms in the HLA D region have been reviewed in
Trowsdale et ah, 1985, Immunol. Rev. 85:5-43,
The polymorphism of the HLA D region gene products has, in general, been
defined by serologic typing reagents and by the mixed lymphocyte culture (MLC) reaction
in which T cell proliferation in response to hornozygous lyping cells (HTC) is measured in
culture. The HLA DP antigens were originally defined by their ability to stim~ te a strong
secondary response in specifically primed T cells, a method known as primed lymphocyte
typing (PLT) and described by Mawas et al., 1981, Tissue Antigens 15:458 466; Want et
ah, 1978, Immunogenetics 6:107-115; and Shaw et ah, 1980, J. ~. Med. 152:565 580.
The HLA DP ant1gens elicit only a wea~ response in a ~ J ~LC, and u~like the studies
of HLA DR and ~lLA DQ polymorphism, the analysis of allelic variaaon iD d~e HLA DP - -
region has been co~li~3~ y ~e lack of availa~ y of ser~logic ~.,age.~ls and o~ typing
~ 133920G
cells. Ln addition, the specific cell lines used in the PLT assay are difficult to generate, and
the typing assay is slow and somewhat variable from laboratory to laboratory.
Cellular (see Odum et aL, 1987, Tissue Antigens 29:101-109), biochemical (see
Lotteau et ah, 1987, Immunogenetics 25:403-407), and restriction fragment lengthpolymorphism (RFLP, see Hyldig-Nielsen et ah, 1987, Proc. Natl. Acad. Sci. USA
84:1644-1648) analyses have indicated that the degree of polymorphism in thé DP region
may be more extensive than the currently serologically, immunologically, or PLT-defined
DPwl through DPw6 types. The RFLP approach showed that the RFLP of the DP regionwas relatively extensive and that some of the fr~ mÇntc were strongly ~csQci~ted with
certain DP antigens. However, the RFLP technique has certain limit~t-ions. An allele
carrying a variant sequence is identifiable only if the variant nucleotide is within the
recognition site of a restriction enzyme used in the analysis or if a polymorphic restriction
site is in linkage disequilibrium with a specific coding sequence variation. In addition,
RFLP analysis simply provides evidence that a coding sequence variation exists but does
not provide information on the exact nature of the variation. Moreover, relatively large
fragments of the genomic nucleic acid must be used for the analysis. This latter~ui~ ent often rules out the use of samples which have been kept under conditions
which result in the degradation of the genomic DNA.
Accurate DP typing may prove hll~ol L~t in several m~lic~l applications. Geneticrecombination between the DP loci and the serologically typed DR loci can occur such that
serologically identical sibs may not be matched at DP. The HLA DP differences have been
revealed by RFLP analysis in several cases of acute graft vs. host disease between
appa,ently HLA-identical donor and recipient pairs, as described by Amar et aL, 1987, J.
Immunolo~y 138: 1947-19S3. Furthermore, several autoimmune ~ e~ces, including
coeliac di~e~e, have been shown to be associated with specific DP types, defined either by
PLT, Odum et_., 1986, Tissue Antigens 28:245-250, or by RFLP, Howell et al., 1988,
Proc. Natl. Acad. Sci. USA 85:222-226, analysis.
A ~ignific~rlt irnprovement in DNA ~mplification, the polymerase chain reaction
(PCR) technique, was disclosed by Mullis in U.S. Patent No. 4,683,195, and methods for
utilizing PCR in the cloning and detection of nucleic acids were disclosed by Mullis I aL in
U.S. Patent No. 4,683,202. In the PCR technique, short oligonucleotide primers are pl~cd
to match opposite ends of the sequence to be amplified. The sequence between the primers
need not be known. A sample of nucleic acid (DNA or RNA, although RNA is first
_v
133920~
converted to cDNA in the PCR process) is extracted and denatured, preferably by heat, and
hybridized with oligonucleotide primers which are present in molar excess. Polymerization
is catalyzed by a polymerase in the presence of deoxynucleotide tnphosphates (dNTPs).
This results in two "long products" which contain the respective primers at their 5'-termini,
covalently linked to the newly synthesized complements of the original strands. The
replicated DNA is again denatured, hybridized with oligonucleotide primers, returned to
polymerizing conditions, and a second cycle of replication is initiated. The second cycle
provides the two original strands, the two long products from cycle 1 and two long
products of cycle 2, and two "short products" replicated from the long products produced
in cycle 1. The short products contain sequences (sense or antisense) derived from the
target sequence and flanked at the 5' end with a primer and at the 3'-end with a sequence
complementary to a primer. On each additional cycle, the number of short products is
replicated exponentially. Thus, the PCR process causes the amplification of a specific
target sequence and allows for the detection of sequences initially present in a sample in
only extremely small amounts.
Allelic sequence variations in the gene encoding beta-globin of hemoglobin and in
the gene encoding HLA DQalpha have been detected by utilizing allele-specific
oligonucleotides (ASO), which will only anneal to sequences that match them perfectly, as
described by Saiki et ah, 1986, Nature 324:163-166. These studies also utilized the PCR
procedure to amplify the DNA sequences present in the samples and a dot-blot technique to
detect probe hybridization to the sample.
The present invention solves the above listed problems in HLA DP typing and
provides a relatively rapid, convenient, practical, accurate, and reproducible method for
determining HLA DP genotypes. The method is based in part upon the finding that there
are a large number of previously unreported DPbeta alleles. The variation between alleles
is of a highly dispersed nature. The present invention also provides nucleic acid probes for
the regions of the HLA DPbeta gene that are most informative in discrimin~ting between
different DP alleles for use in the method.
These oligonucleotide probes are called SSOs (sequence specific oligonucleotides),
which, under the appropriate conditions, are able to bind specifically to their
complementary sequences. If a particular probe can be used to identify uniquely an allele,
the probe is called an al~ele specific oligonucleotide (ASO). Because of the dispersed
nature of the variation between the DPbeta alleles, rarely is any one probe able to identify
uniql~ely a specific DPbeta allele. Rather, according to the methods of the present
133920~
invention, the iden~ty of an allele is inferred from the pattern of binding of a panel of
probes, with each individual probe of the panel specific for different segments of the HLA
DP gene. The probes comprise a nucleotide sequence totally complementi~ry to a variant
sequence of a variable segment of a DP gene. The complementary sequence of the probes
is usually from 10 to 30 nucleotides in length, most often 17 to 19 nucleotides in length.
As noted above, PCR is quite an important tool in nucleic acid based diagnostic
methods. The novel probes of the invention can of course be used to detect specific
sequences in PCR-amplified DNA. To enable practice of PCR-based, HLA DP DNA
typing, the present invention provides oligonucleotide primers that allow for the
amplification of informative DP regions via PCR.
Accordingly, one aspect of the invention is a process for determining an
individual's HLA DP genotype from a nucleic acid containing sample obtained from the
.
lndlvldual comprlslng: (a) ampllfylng a target reglon of sa1d nuclelc ac1d whlch contauns a
polymorphic region of an HLA DP gene; (b) hybridizing the amplified nucleic acid with a
panel of sequence specific oligonucleotide (SSO) probes specific for variant segrnents of
HLA DP genes under conditions that allow said SSO probes and amplified nucleic acids to
form stable hybrid duplexes; and (c) detecting hybrids formed between the amplified
nucleic acids and the SSO probes.
Another aspect of the invention relates to kits useful for determining the HLA DP
genotype of an individual; these kits comprise a panel of SSO probes for allelic variant
sequences in said target region; and (b) instructions for determining the genotype by
utilizing kit ingredients.
To aid in understanding the invention, several terms are defined below.
"Genotype" refers to a description of the genotypic variants of an allele present in
an individual (or sample). The genotype of an individual may be determined, for example~
by the use of serologic reagents, by the use of typing cells, by RFLP analysis of the
genome, and, as discussed below, by the use of SSOs.
"HLA DP region" or "DP" refers to that region of the HLA D complex, described inTrowsdale et ah, supra, which lies on the centromeric side of the HLA D region and
contains two alpha and two beta genes, one pair of which are pseudogenes.
"Oligonucleotide" refers to primers, probes, nucleic acid fragments to be detected,
nuc~eic a~;ià controls, and unlabeled blocking oligomers and is defined as a molecule
c~nprised of two or more deoxyribonucleotides or ribonucleotides. The exact size of an
oligonucleotide will depend upon many factors and the 1lltim~te function or use of the
~ ~ 1339206
oligonucleotide. Oligonuc~eotides can be prepared by any suitable method. Such methods
include, for example, cloning and restriction of a~ploy,iatc sequences and direct çhemic~l
synthesis by a method such as the phosphotriester method of Narang et al., 1979, Meth
Enzymol. 68:90; the phosphodiester method of Brown et aL, 1979, Meth. Enzymol.
68:109; the diethylphosphoramidite method of Beaucage et aL, 1981, Tetrahedron Lett.
22:1859-1862; and the solid support method in U.S. Patent No. 4,458,066.
"Polyrnorphic" or "DNA polymorphism" refers to the condition in which two or
more ~ ;alions of a specific DNA sequence coexist in the same inlu'~l~;cding population.
"Primer" refers to an oligonucleotide, whether natural or synthetic, capable of
acting as a point of initiation of DNA synthesis under con~lition~ in which synthesis of a
primer extension product comple, - ,e~ y to a nucleic acid strand is in~uce~ ~ i.e., in the
presence of four differentnucleoside triphosphates and an agent for polymerization (i.e.,
DNA polymerase or reverse transcriptase) in an ap~ulo~liate buffer and at a suitable
le"~,ature. A primer is preferably an oligodeoxyribonucleotide and is single stranded for
maximum efficiency in amplification, but may also be double stranded. If double stranded,
the primer is first trsated to separate its strands before being used to prepare extension
products. The exact length of a primer will depend on many factors, but typically ranges
from 15 to 2S nucleotides. Short primer molecules generally require cooler l~ c.~tures to
form sufficiently stable hybrid complexes with the template. A primer need not reflect the
exact sequence of the template but must be sufficiently comple,~ . y to hybridize with a
template. An example of a non-complementary sequence which may 'oe incorporated into
the primer is a sequence which encodes a restriction enzyme recognition site (see U.S.
Patent No. 4,800,159~.
"Primer," as used herein, may refer to more than one primer, particularly in the case
where there is some ambiguity in the infol malion regarding one or both ends of the target
region to be amplified. For inct~nce, if a nucleic acid sequence is inferred from a protein
se~uence, a "primer" is actually a collection of prirner oligonucleotides co~ il-g
sequences representing all possible codon variations based on the degeneracy of the genetic
code. One of the pnmer oligonucl~o~id~s in this collection will be homologous with the
end of ehe target sequence. Likewise, if a "conserved" region shows signific~nt levels of
polymorphism in a population, mixtures of primers can be prepared that will arnplify
-~j~t se~uences. A primer can be labeled, if desired, by incorporating a label detectable
by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For
example, useful labels include 32p, fluurescel~dyes, cl~f[~n-dense l~nt~ enzymes (as
B
133~20~
commonly used in ELISAS), biotin, or haptens or proteins for which antisera or
monoclonal antibodies are available. A labels can also be used to "capture" the primer, so
as to facilitate the immobilization of either the primer or amplified DNA on a solid support.
"Restriction endonucleases" and "restriction enzymes" refer to enzymes, usually of
bacterial origin, that cut double-stranded DNA at or near a specific nucleotide sequence.
"Restriction fragment length polymorphism" or "RFLP" refers to the differences in
DNA nucleotide sequences that are randomly distributed throughout the entire genome and
that produce different restriction endonuclease patterns for dirre~ t individuals upon
digestion of genomic DNA.
"Sequence-specific oligonucleotide" or "SSO" refers to oligonucleotides that have
an exactly comple,.l~r,la.y sequence to the sequence to be detected, typically sequences
characteristic of a particular DP allele, which under "sequence-specific hybridization
conditions will hybridize only to that exact comple.,le,.l~uy target sequence. The
hybridization is under stringent conditions. Stringent hybridization conditions are known
in the art, and are described, for example, in Maniatis et aL, Molecular Cloning: A
Laboratory Manual (New York, Cold Spring Harbor Laboratory, 1982). Depending on the
sequences being analyzed, one or more sequence-specific oligonucleotides may be
employed for each sequence. The terms "probe" and "SSO probe" are used
interchangeably with SSO.
"Target region" refers to a region of a nucleic acid which is to be analyzed, which
usually contains polymorphic DNA sequences.
"Therrnostable polymerase enzyme" refers to an enzyme which is relatively stable to
heat and which catalyzes the polymerization of nucleotides to form primer extension
products that are complementary to one of the nucleic acid strands of the target sequence.
Generally, the enzyme will initiate synthesis at the 3'-end of the target sequenr,e ~ltili7ing
the primer and will proceed in the S'-direction along the template until synthesis tenninates
A purified therrnostable polymerase enzyme is described more fully in I Ul~)e~ll Patent
Office (EPO) Publication 258,017 and is cornmercially available from Perkin-Elmer Cetus
Instrllmrnt~. '
The present invention provides processes and reagents for dele. "~ ing the HLA DP
genotype of an individual. In part, the invenhon results from dle discovery o-f
polyn~Tphisms in the variable second exon of the HLA DPbeta genes by d~e combined
methods of PCR amp~ifir~hon, cloning, ar.~d DNA sequencing. As a result, 22 DPbeta
(DPB) allelic variants have been ~sco~le~d. Based up~n the nove~ sequence of these DP
133920~
genes, SSO probes are provided for the detection of the variant genotypes. The variations
between the different DP alleles are dispersed. Therefore, one probe alone is rarely able to
identify uniquely a specific DPbeta allele. Rather, the identity of an allele is inferred from
the pattern of binding of a panel of probes, with each individual probe of the panel specific
to different segments of a DP gene.
In a preferred embodiment of the invention, the process for DNA based typing of
HLA DP genotypes is comprised of amplifying a nucleic acid sequence which contains a
variable portion of an HLA DP gene, determining the variant HLA DP sequence present
using SSO probes; and inferring the HLA DP genotype from the pattern of binding of the
l o SSO probes to the amplified target sequence. To facilitate practice of this preferred
embodiment, the present invention provides primers useful in amplifying by PCR the HLA
DP target region.
In this preferred method, a sample containing nucleic acid is obtained from an
individual whose HLA DP genotype is to be determined. Any type of tissue containing
HLA DP nucleic acid may be used for purposes of the present invention. Because the
present invention also is compatible with amplified nucleic acids, and because the PCR
technique can amplify extremely small quantities of nucleic acid, samples containing
vanishingly small amounts of nucleic acid can be typed for the presence of particular HLA
DP variants by the method of the present invention. For instance, even a single hair,
contains enough DNA for purposes of the present invention, as evidenced by the work
with DQalpha described by Higu~hi et ah, 1988, Nature 332:543-546.
In general, the nucleic acid in the sample will be DNA, most usually genomic DNA.
However, the present invention can also be practiced with other nucleic acids, such as
messenger RNA or cl~ned DNA, and the nucleic acid may be either single-stranded or
double-stranded in the sample and still be suitable for purposes of the present invention.
Those of skill in the art recognize that whatever the nature of the nucleic acid, the nucleic
acid can b~ typed by the present method merely by taking appropriate steps at the relevant
stage of the process. If PCR is used to amplify the nucleic acid in the sample, then the
sample will usually comprise double-stranded DNA when typed with the novel probes of
the invention.
As noted above, in a preferred embodiment, the HLA DP typing method and probes
of the invention are used in conjunction with PCR-amplified target DNA. Those practicing
the present inventi~n shou~d note, lu)wever, that amplification of HLA DP target sequences
in a sample may be accomphshe~ by any known method which provides sufficient
13392~
amplification so that the target se~uence may be detected by nucleic acid hybridization to an
SSO probe. Although the PCR process is well known in the art (see U.S. Patent Nos.
4,683,195 and 4,683,202) and although a variety of commercial vendors, such as Perkin-
Elmer/Cetus instruments, sell PCR reagents and publish PCR protocols, some general
PCR information is provided below for purposes of clarity and full understanding of the
invention to those unfamiliar with the PCR process.
To amplify a target nucleic acid sequence in a sarnple by PCR, the sequence mustbe accessible to the components of the amplification system. In general, this accessibility is
ensured by isolating the nucleic acids from the sample. A variety of techniques for
extracting nucleic acids from biological samples are known in the art. For example, see
those described in Maniatis et al., Molecular Cloning: _ Laboratory Manual, (New York,
Cold Spring Harbor Laboratory, 1982). Alternatively, if the sample is fairly readily
disruptable, the nucleic acid need not be purified prior to amplification by the PCR
technique, i.e., if the sample is comprised of cells, particularly peripheral blood
lymphocytes or amniocytes, Iysis and dispersion of the intracellular components may be
accomplished merely by suspending the cells in hypotonic buffer.
Because the nucleic acid in the sample is first denatured (assuming the sample
nucleic acid is double-stranded) to begin the PCR process, and because simply heating
some samples results in the disruption of cells, isolation of nucleic acid from the sample
can sometimes be accomplished in conjunction with strand separation. Strand separation
can be accomplished by any suitable denaturing method, however, including physical,
chemical, or enzymatic means. Typical heat denaturation involves temperatures ranging
from about g0~C to 1 05~C for times ranging from about 1 to 10 minutes. Strand separation
may also be induced by a helicase, an enzyme capable of exhibiting helicase activity. For
example, the enzyme RecA has helicase activity in the presence of ATP. The reaction
conditions suitable for strand separation by helicases are known in the art (see Kuhn
Hoffman-Berling, 1978, CSH-Ouantitative Biology 43:63 and Radding, 1982, Ann. Rev.
Genetics 16:405-436).
As noted above strand separation may be accomplished in conjuction with the
isolation of the sample nucleic acid or as a separate step. In addition, strand separation can
be achieved simultaneously with the next step in the PCR process: annealing of primers
and synthesis of primer extension products. In this embodiment of the PCR process, the
temperature is very carefully controlled so that strand sepa}~tion and primer annealing and
extension occur in equilibrium. In this embodiment of the PCR process, the reaction is
--~ ' 1339206
catalyzed by a heat-stable polymerase and carried out at an elevated temperature. The
le,~ dlur~ is one at which the enzyme is thermostable, and at which the nucleic acids are
in an ~quilil),ium ~ single and double strands, so that sufficient primer will anneal to
template strands to allow a reasonable rate of polymerization. In the ~rell~d embodiment
of the PCR process, however, strand separation is achieved by heating the reaction to a
sufficiently high temperature for an effective time to cause the denaturation of the duplex,
but not to cause an irreversible denaturation of the polymerase (see EP No. 258,017).
No matter how strand separation is achieved, however, once the strands are
s~ ed, the next step in PCR involves hybridizing the se~dted strands with primers that
flank the target sequence. The primers are then extended to form complement~ry copies of
the target strands, and the cycle of denaturation, hybridization, and extension is repeated as
many times as necessary to obtain the desired amount of amplified nucleic acid.
As noted above, the present invention provides PCR primers for HLA DP DNA
amplification and typing. These primers are complementary to sequences in the conserved
regions that flank the target sequences in the variant regions of the HLA DP loci. For
purposes of the present invention, the preferred variant region of the HLA DP loci is the
second exon of the DPalpha and DPbeta genes. For successful PCR amplification, the
present primers are designed so that the position at which each primer hybridizes along a
duplex se~uence is such that an extension product synthesi7~d from one primer, when it is
separated from its template (complement), serves as a template for the extension of the
other primer to yield an amplified segment of nucleic acid of defined length. Moreover,
primers are provided that will bind p~c~re~ltially to the HLA DP region under selective
annealing conditions.
Template-dependent extension of primers in PCR is catalyzed by a polymerizing
agent in the presence of adequate amounts of fwr deoxyribonucleo'~ide triphosphates
(dATP, dGTP, dCTP, and drrP) in a reaction medium comprised of the a~l~liate salts,
metal cations, and pH buffenng system. Suitable pol~ g agents are enzymes known
to cataly~ template-dependent DNA synthesis. For example, if the template is RNA, a
suitable polymerizing agent to convert the RNA into a compleme.-~ y DNA (cDNA)
sequence is reverse transcriptase (RT), such as avian myelo~l~ctQsi~ virus RT. Once the
target for amplification is DNA, suitable polyrnerases include, for example, E. coli DNA
polymerase I or its K]enow fragmellt, T4 DNA polymerase, and Taq polymerase, a heat
stable DNA yol~l.~ase isolated from Thennus a~uaticus and co.l~ ;ially available from
Perkin-Efmer~Cetus Inst}uments (PECI). The ~atter enzyme is widely used in the
~ ,
~ 1339206
10 ~,
amplification and sequencing of nucleic acids. The reaction conditions for using DNA
polymerases are known in the art, and are described in, for example, the treatise Methods
in Enzymology, and in Maniatis et al., Molecular Cloning: A Laboratory Manual, supra.
The PCR method can be performed in a step-wise fashion, where after each step
new reagents are added, or in a fashion where all of the reagents are added cimlllt~ne
or in a partial step-wise fashion, where fresh or dirre~ t reagents are added after a given
number of steps. For example, if strand separation is indllced by heat, and the polymerase
is heat-sensitive, then the polymerase will have to be added after every round of strand
separation. However, if, for example, a helicase is used for denaturation, or if a
thermostable polymerase is used for extension, then all of the reagents may be added
initially, or, alternatively, if molar ratios of reagents are of consequence to the reaction, the
reagents may be repleniched periodically as they are depleted by the synthetic reaction.
Those skilled in the art wi!l know that the PCR process is most usually carried out as
an auloll,aled process with a thermostable enzyme. In this process, the reaction ll)i~CIUl'~ iS
cycled through a denaturing region, a primer annealing region, and a reaction region. A
machine specific~lly adapted for use ~vith a ther nostable enzyme is disclosed more
completely in EP 236,069 and is commercially available from PECI.
One reason, as noted above, the PCR process is important in the method of the
present invention is that the PCR process can be used to amplify the sample nucleic acid
prior to HLA DP DNA typing. Another important use of PCR for purposes of the.present
invention, however, is for determining the nucleotide sequence of previously undiscovered
allelic variants which exist in the HLA DP region, so that probes for those variants can be
constructed and used in the present method. In this use of the PCR process, polymorphic
regio~s of the DPalpha and DPbeta genes are amplified, and the nucleotide sequences of
these polymorphic target regions, for example, the second exon of the DPalpha and DPbeta
genes, are deterrnined. As illustrated below, it is also useful for the cells containing a
particular variant to be typed by serological typing, mixed Iymphocyte typing, or primed
lymphocyte typing to correlate the nucleotide sequence of a particular variant with the DP
type established by prior art methods.
Analysis of the nucleotide sequence of the target region of a DP variant allele can be
readily perforrned by direct analysis of the PCR products. A ~,leÇelled sequencing protocol
is described by lnnis et al., 1988, Proc. Natl. Acad. Sci. 85:9436-9440. --
A process for direct sequence analysis of PCR amplified products is also described
by Saiki et al., 1988, Science 239:487-491. Alternatively, the amplified - -
B .
~ 1339206
target sequence may be cloned prior to sequence analysis, as described by Scharf et ah,
1986, Science 233:1076-1078.
As di~cus~ed in the Examples below, a panel of DPw typed cells, ~ esen~;ng a
large number of different haplotypes, has been analyzed by PCR and nucleotide
sequencing of the second exon of the DPbeta gene. As a result of this effort and similar
efforts using samples obtained from a variety of individuals suffering from aulo;.-----.lne
dice~es, tli~cussed in greater detail below, 22 dirfele,-t allelic variants in this locus were
discovered. In general, the results del"orl~L,a~d that specific DPbeta sequences correlate
with the standard PLT-defined DPwl through DPw6 speçificjtiçs. The rare exceptions
may reflect the difflculty of obtaining a sl~ldald and ~e~n)du-,ible PLT DPw typing system,
which difficulties highlight the advantages of the present method. In this regard, it is
relevant that the cell line Cox, originally typed as DPwl, has recently been retyped as
DPw3 and contains the DPB3 allele.
Other~interesting and important correlations between the serologically defined DP
types and DP variant nucleotide sequences have been discovered as a result of the advances
provided by the present invention. For instance, the DPw4 type of the prior art can now be
- subdivided into two DPbeta subtypes (designated DPB4. 1 and DPB4.2) by the method of
the present invention. The DPB4.2 allele was found in two (APD and LBI) DPw4
homozygous typing cells (HTCs) known to have an unusual DPw4 typing. DPbeta
subtypes (designated DPB2. 1 and DPB2.2) for the DPw2 type were also discovered as a
result of the present invention.
Yet another significant advance provided by the present invention relates to thediscovery of a large number of variant DPbeta alleles in a variety of cells that type by the
prior art methods as DP "blank." These variant alleles have been designated with DPB
numbers of 7 and above. Further analysis of DPw blank haplotypes will probably lead to
the identification and characterization of additional DPbeta alleles that can ~en be typed by
the present nlethod DPw blank cells are those which fail to stim~ tç the eYi~ting PLT
panels of T cell reagents. Unlike the serologic DQw blank specificity, which, as described
by Horn et al., 1988, Proc. Natl. Acad. Sci. USA 85:6012-6016, is encoded by a unique
DQB sequence, the DPw blank specificity is heterogeneous. Because the haplotypicfrequency of the DPw blank is estim~ted to be 40~o, the present invention is especially
important in that it for the first time enables the ~ g of this large DPw type.
The availability of DPbeta DNA sequences from DPw typed cells not only makes
possible the subdivisi~n of the se.ol~gi~al DPw types but also makes possible the - -
~,....................................................................... .
13392~6
identification of the Ei~ear epltopes defined by serologic and cellular typing reagents. For
example, the antibody SP42 reacts with DPw2 and DPw4 cells. The only sequence unique
to these two specificities is the GlyGlyProMet (GGPM) at positions 84 to 87, which are
therefore believed to constitute the SP42 epitope. Likewise, the antibody SP3 reacts with
DPw3 and DPw6 cells, as well as with some additional cells like the DPw bla~k lines Tok
and Akiba. These lines bear the specificity Cp63 and contain the closely related DPbeta
alleles DPB9 and DPB 12. The only sequence unique to SP3 reactive cells is the acidic
AspGluAsp (DED) sequence at positions 55 to 57. The SP3 epitope therefore seems to
map to this region of the DPbeta chain.
As yet another e~a~ ulc of how the novel DP sequçnces of the invention allow themapping of serologically defined epitopes, the antibody DP11.1 reacts widl DPw2 and
DPw4 cells. Bodmer et ah, 1987, Proc. Acad. Sci. lJSA 84:4596-4600, showed with
Western blot analysis that this antibody binds to the DPalpha chain. Given the pattern of
DPbeta polymorphisms provided by the present invention, this observation suggests that
the DP 11.1 antibody binds to a confonnational determinant formed by the DPalpha chain
and DPbeta chains containing the GlyGlyProMet (GGPM) sequence at positions 84 to 87.
Polymorphic residues in the HLA DP region may also constitute ~ilo~es
recognized by T cell clones. Cells from clone 1666 react with DPw5 and some DPw2cells. ABL (DPB2.2) is positive and WPV (I)PB2.1) is negative, as disclosed at the Tenth
International Histocompatibility Workshop. These C1666 cells may recognize the
GluAlaGlu (EAE) residues at position 55 to 57, because this sequence is unique to the
DPB2.2 and DPB5 alleles. A Leu (as opposed to Phe) residue at position 35 is also unique
to C1666 reactive cells.
The same approach of correlating specific polymorphic residues with DPw
specificities can be used to map the epitopes defined by PLT typing. For example, the only
difference between the DPB2.1 and DPB4.2 alleles is the Glu to Lys change at position 69.
Because cells containing these alleles are distinguished from each other in the PLT system,
the charge of the polymorphic position 69 residue is believed to be involved in the epitope
recognized by P~T typing celis Similarly, the only differences between the DPB3 and
DPB6 alleles is the Lys to Glu substitution at position 69, as well as Val to Met change at
posi~ion 76.
As illustrated by the foregoing, the novel DPbeta sequences provided by the present
invention not only provide the basis for a very useful DPbeta DNA typing method but
provide much useful information in the int~y'c~d~i~n of serological DPbeta typing results. - -
~ .
1339206
13
The pre~ent invention also enables one to perforrn HLA DNA typing not only on the
DPbeta locus but also on the DPalpha locus as well. As illustrated below, at the present
time, only two variant alleles are known to exist at the second exon of the DPalpha locus
(see Trowsdale et ah, supra). The protein sequences encoded by the second exon of these
two alleles (these alleles are designated DPA l and DPA2) differ from each other by only
three amino acids, and these differences do not appear to correlate with any specific PLT-
defined type. Thus, the DPalpha variation is much less extensive than predicted by the
RFLP variation detected with DPalpha probes (see Hyldig-Nielsen et al., 1987, Proc. Natl.
Acad. Sci. USA 84: 1644- 1648), suggesting that most of the polymorphisms detected in
the DPalpha gene by RFLP analysis are either in noncoding sequences or in a nearby
DPbeta locus. The observed correlations between DPw types and DPalpha RFLP markers
therefore probably reflects linkage disequilibrium between DPbeta sequence variation and
the polymorphic restriction sites cletected with the DPalpha probe, as does the association
between DRalpha RFLP markers and DR specificities (see Stetler et ah, 1985, Proc. Natl.
Acad. Sci. USA 82:8100-8104).
The DNA sequences of the DPalpha and DPbeta genes serve as a useful starting
point in the design of the sequence specific oligonucleotide probes of the present invention.
These probes are designed so that, under stringent hybridization conditions, the probes
hybridize specifically only to exactly complementary sequences in variant segments of the
DPalpha and DPbeta alleles. These SSO probes may be of any length which spans the
variant sequences in a variant region and allows for sequence specific hybridization, but
preferably the hybridizing region of the probe is short, in the range of 10 to 30 bases, and
more preferably is about 17 to 19 bases in length. For immobilization, the probe may also
contain long stretches of poly T which can be fixed to a solid support by irradiation.
The SSO probes of the invention are also designed to hybridize specifically with a
particu~ar variant segment of a DP allele and to have destabilizing mismatches with the other
variant sequences known for the particular segment. Preferably, the probes are specific for
variant DNA segments in the variable second exons of the DPalpha and DPbeta genes, and
even more preferably, the probes are specific for DNA segments encoding the residues near
positions 8-11, 36, 55-57, 65-69, 76, and 84-87 of the second exon. Oligonucleotide
probes which h~ve been designed to hybridize specifically to the second exons of the
DPbeta ~nd DPalpha a~leles ar~ de~nbed in more detail below and in the Examples.The probes of the invention can be s~n~hesized and labeled using the techniques
described above in the discus.sion o~ PCR primers. For example, the probe may be labeled
~''--''1 ' , 133S2~6
14
at the 5'-end with 32p by incubating the probe with 32P-ATP and kinase. A suitable non-
radioactive label for SSO probes is horseradish peroxidase (HRP). Methods for preparing
and detecting probes containing this label are described in the Examples below. For
additional information on the use of such labeled probes, see U.S. Patent No. 4,789,630;
Saiki et aL, 1988, N. ~g. J. Med. 319:537-541; and Bugawan et al., 1988,
Bio/Technology 6:943-947. Useful chromogens include red leuco dye and T~IB.
The probes of the invention can be used to identify the allelic sequences present in a
sample by determining which of the SSO probes bind to the HLA DP sequences present in
the sample. Suitable assay methods for purposes of the present invent-ion to detect hybrids
formed between SSO probes and nucleic acid sequences in a sample are known in the art.
For example, the detection can be accomplished using a dot blot format, as described in the
Examples. In ~he dot blot format, the unlabeled amplified sample is bound to a membrane,
the membrane incubated with labeled probe under suitable hybridization conditions,
unhybridized probe removed by washing, and the filter monitored for the presence of
bound probe. When multiple samples are analyzed with few probes, a plefe-l.,d process
requires high stringency hybridization and wash conditions, allowing only perfectly
matched hybrids to exist.
An alternative method is a "reverse" dot blot format, in which the amplified
sequence contains a label. In this format, the unlabeled SSO probes are bound to the
membrane and exposed to the labeled sample under ~p~liately stringent hybritli7~ion
conditions. Unhybridized labeled sample is then removed by washing under suitably
stringent condilions, and the filter is then monitored for the presence of bound sequences.
In another version of the "reverse" dot blot format, the SSO probe is labeled, and the
sample nucleic acid is unlabeled. After hybridization and washing, the labeled probe, or a
labeled fragment of the probe, is released from the "~I"bl~ne and detected to d~ ? if a
sequence in the sample hybridized to the labeled oligonucleotide Release of label can be
achieved by digestion with a restriction enzyme that recognizes a restriction site in the
duplex hybrid. This procedure, known as oligomer restriction, is described more fully in
U.S. Patent No. 4,683,194 and corresponding EP Patent Publication 164,054,
Whatever the method for de~ inil g which DP SSO probes of the inv~ntion
hybridize to DP sequences in a sample, the central feature of the DP DNA typing method
involves the identification of the HLA ~P alleles present in the sample by analyzing the
~ 1339206
pattern of bindin~ of a panel of SSO probes. Although single probes of the invention can
certainly be used to provide useful information, the variation in the DPbeta alleles is
dispersed in nature, so rarely is any one probe able tO identify uniquely a specific DP
variant. Rather, as shown in the Examples, the identity of an allele is inferred from the
pattern of binding of a panel of SSO probes, which are specific to different segments of the
DPalpha and DPbeta genes.
DNA typing of HLA DP alleles is useful for many different purposes. For
example, DPbeta polymorphism is involved in tissue rejection of allografts. Bone marrow
transplantation studies revealed that three apparently HLA-identical donor-recipient pairs
who exhibited acute graft versus host disease and weak MLC reactivity were, by RFLP
analysis, all different in the DP region, as described by Amar et al., J. Immunolo~y
138:1947. Th~ls, useful methods for preventing graft rejection will involve matching the
HLA DP types of the donor and recipient by applying the present method of HLA DP DNA
typing: inferring the HLA alleles present in donor and host from the pattern of
hybridization of SSO probes to DPbeta and/or DPalpha nucleic acid sequences present in
samples obtained from both donor and host.
Another important application for the present method of HLA DP DNA typing is in
determining an individual's susceptibility to autoimmune diseases linked to certain HLA DP
alieles. The association of particular DP alleles with an autoimmune disease can be
discovered by using the present method to determine the frequency with which a given
allele is present in individua5s suffering from the autoimmune disease and comparing that
frequency with the frec~uency determined for healthy individuals of a control group, as has
been done with other HLA typing systems. For example, Howell et ah, 1988, Proc. Natl.
Acad. Sci. USA 85:222-226, reported a significant association between coeliac disease
(CD) and a pair of HLA DPalpha and HLA DPbeta RFLPs. Such studies often provide the
additional benefit of the discovery of previously uncharacterized DP alleles, in both patient
and control groups. For instance, the DPB l 3 through DPB 19 alleles, the sequences of
which represent an important aspect of the present invention, were discovered in the course
of studies involving autoimmune disease patients and control groups.
In just such a fashion, an initial study of four CD patients who were typed by the
method of the present invention revealed that the DPB4.2 allele confers CD susceptibility.
A more extensive study, based on samples from Italian individuals, also showed that the
calculated relative risk (RR~ of CD for the DPB4.2 allelle is 9.3 and that six of the eleven
CD patients typed by the prese~nt method calTied the DPB3 allele. About 78% of CD
~ 133920~
16
patients examined by the present method carry either the DPB4.2 or the DPB3 alleles, so
that the RR for the presence of either of these alleles is 13.5. In yet another study of U.S.
individuals with CD, an increase in the DPB1 allele frequency, as well as an increase in the
DPB4.2 allele frequency, was observed when compared to the frequency of these alleles in
a control group. Such studies also revealed that specific combinations of DQbeta alleles
(DQB2), DQalpha alleles (DQA4) and DPbeta alleles (DPB4.2 or DPB3) are associated
with increased risk of CD susceptibility.
As shown from the foregoing, the present invention provides important new
methods for determining the CD susceptibility of an individual, which methods comprise
determining whether an individual carries a DP allele associated with CD susceptibility. As
shown herein, such CD susceptibility-conferring alleles include the DPB4.2, DPB3, and
DPB I alleles, and as DPB4. 1 is a relatively common allele, there is an increased frequency
of the genotype DPB4. 1/4.2 in CD susceptible individuals.
As with CD, the present invention provides similar advances with respect to other
serious autoimmune diseases. For instance, studies that correlate allelic frequency with
disease show that the frequency of specific DPbeta alleles appears to be higher in insulin
dependent diabetes mellitus (IDDM) and myasthenia gravis (MG) patients than in otherwise
HLA matched controls. For MG, the frequency of DP alleles that code for the sequence
DEAV at the 3' end of the second exon, such as DPB3 and DPB5, is increased. In
addition, the frequency of the DPB4. 1 allele in MG patients (relative to healthy control
individuals) is dramatically decreased, suggesting that DPB4.1 may conferprotection from
MG. For IDDM, the frequency of DPbeta alleles DPB2. 1, DPB 1, DPB4. 1, and DPB 13 is
increased. Many of the ~DDM patients studied that carried the DPB13 allele also carried the
HLA alleles B18 and DR3.
The DP typing methods of the present invention have also provided important
advances in determining whether an indi~idual is susceptible to certain forms of arthritis.
For instance, patients with classical rheumatoid factor positive adult rheumatoid arthritis
(ARA) show an increased frequency of the DPB4.2 allele, although more studies must be
completed to ensure that this increase is statistically significant. In addition, scientists have
long known that the DPw2 serologic type is associated with susceptibility to pauciarticular
juvenile rheumatoid arthritis (JRA~. As noted above, the present invention has for the first
time allowed the sero)o~c DP~2 type to be su'odivided into two DPB DNA types, and the
disease-susceptibility ty~ing methods of t~e p~esent invention demonstrate that the known
DPw2-associated susceptibility to JRA is more specifically attributable to the DPB2. 1
133 9 2 0 ~
allele. About 55% of the JRA patients examined were positive for DPB2. 1, while only
16% of control individuals had this allele, giving a RR of 6.3 for JRA in individuals with
the DPB2. l allele. The association of JRA with the DPB2. l allele is independent of
linkage with previously defined HLA DP region markers, and the significance of this
DPB2. 1 association with JRA can be more readily appreciated when one considers that the
DPB2. 1 sequence differs from the DPB4.2 allele (apparently not associated with JRA
susceptibility) by only one amino acid at position 69 of the B 1 domain. It should be noted,
however, that most JRA patients with the DPB2. 1 allele also had a disease-associated DR
marker such as DRw8, DR5, or DRw6.
Thus, the present invention also provides a method for detecting JRA susceptibility
that comprises treating HLA DP genomic DNA of an individual, preferably after
amplification, with oligonucleotide probes specific for the DPB2.1 allele and determining if
hybridization has occurred. General practice of the invention will, however, utilize the full
panel of DPbeta probes. As noted above. the present method will be useful in determining
the DP association with a wide variety of autoimmune diseases, not just JRA. This fact can
be more readily grasped by considering the Table below, in which the allelic frequency
(number of alleles of the indicated DP type divided by total number of DP alleles present
and then multiplied by l ()()) for the various DP alleles in a number of patient groups and a
control group is shown. In the Table, MS indicates multiple sclerosis patients.
133920~
18
HLA-DP ALLELIC FREQUENC~ES IN SE~ECTED PATIENT GROUPS
AlleleControls (n=50)MG (n=23)JRA (n=44) MS (n=21~ ARA (n=23)
6% 9% 3% 2% 5%
2. 1 10% 6% 30% 12% 12%
2.2 0% 2% 0% 6% 0%
3 6% 13% 11% 17% 11%
4. 1 56% 25% 40% 48% 41%
4.2 10% 10% 6% 6% 22%
2% l 0% 0% 0% 2%
6 2% ()~ 2% 0% 2%
7 0% 0~7G 0% 2% 0%
8 0% 6~/G 0% 0% 0%
9 0% 6% 1 % 0% 0%
1% 6% 1% 0% 2%
1 l 3% 2% 1% 0% 0%
12 0% 0% 0% 0% o%
13 l'~c '~C/c 1% 2% 0%
l 4 2% 0~c 0% 2% 2%
0% 0% 2% 0% 2%
l 6 0% 0% 1 % 2% 0%
17 1% 2% 0% 0% 2%
1 8 ~% ~%
19 0% 0% 0% 0% 0%
2Q t)% 2% 0% 0% 0%
n = number of individuals typed; 2n is the number of chromosomes tested; controls were
individuals from Northern California.
In general, these studies of autoimmune disease suggest that the charge of
polymorphic residues at positions 55, 56, and 69 of the DPbeta chain are involved in
genetic susceptibility to autoimmune disease. For instance, the change at position 55 from
alanine to aspartate occurs in both the DPB2. 1 and DPB4.2 alleles; however, in IDDM,
which appears to be linked to DPB2. l, there is also a change in the amino acid at position
69. It should be noted, though, that is is the enbre allele, and not simply an amino acid at
one position in an allele, which appears to confer susceptibility.
133920~
19
The effect of the HLA DP alleles on autoimmune disease susceptibility reflects
similar findings in the HLA DR and HLA DQ loci. For instance, Scharf et al., 1988,
Proc. Natl. Acad. Sci. USA 85:3504-3508, and Horn et al., 1988, Proc. Nath Acad. Sci.
USA ~5:6012-6016, report that polymorphic residues at position 57 of the DQbeta chain
5 and position 70 of the DRbeta I chain are implicated in genetic susceptibility to the
autoimmune diseases pemphigus vulgaris (PV) and IDDM.
As noted above, it is anticipated that as medical technology develops, more disease
or disease-prone states, including Grave's disease, systemic lupus erythromatosis, and
Sjogren's Syndrome, will become known to be associated with various DP alleles. The
10 present invention provides methods for distinguishing such alleles from other alleles and so
provides a means to identify individuals at high risk for an autoimmune disease. In a
preferred embodiment, an individual whose susceptibility is to be determined is analyzed
for HLA DP type first by using the PCB method to amplify the target region of the HLA
DP locus. Then, SSO probes are hybridized to the amplified target region, and the
15 particular DP allele present in the amplified DNA is determined from the pattern of
binding of the SSO probes. Finally, one determines whether the allele present in the
amplified DNA is an allele associated with the autoimmllne disease.
The present method, however, is not limited to the field of medical science in
ability to provide significant benefits. DNA typing methods also now play a significant
20 role in the important area of individual identification, whether for solving crimes, as when
the identity of a criminal or victim is established by linking an individual with evidence
left at the scene of a crime, or for solving other issues of a non-criminal nature, as when
biological material is used to cleterrnine the maternity or paternity of an individual.
Whatever the purpose for which the present invention is employed, the diLl~lellces
25 between various DP alleles is key to the success of the method. The most significant
differences between DP alleles can be detected quite readily when the various amino acid
sequences encoded by the alleles are aligned and examined. Such an alignment is shown
below in Table I, where a dash indicates identity with the DPB4.1 allele (for the various
DPbeta alleles and the DPbeta pseudogene, designated SXB) or with the DPA1 allele (for
30 the DPalpha DPA2 allele and the DPalpha pseudogene, designated SXA). In this
depiction, the numbered positions are for the mature peptide subunits, allele designations
are at left, and representative cell sources are at right.
X
TABLE
DPalpha Alleles
DPAl: DHVSTYAAFVQTHRPTGEFMFEFDEDEMFYVDhDKKETVWHLEEFGQAFSFEAQGGLANIAILNNNLNTLIQRSNHTQATN (LB)
DPA2: ---------------------------Q------------------R--------------------------------A- (Daudi)
(SXA): -------E-------S--Y------E-Q---N--E--M--P-P--IHT-D-G--R-I-G-VMARKH---R-NG KQ-W--D
DPbeta Alleles
DPB4.1 NyLFQGRQEcyAFNGTQRFL~KylrNKEEFARFDsDBGEFRAvTELGRpAAEywNsQKDILEEKRAvpDRMcRHNyELGGpMTLQRR (HHK)
DPB4.2 ----------------------------V------------------DE---------------------------------- (APD)
DPB2.1 ____________------------------V------------------DE------------E----------------------- (LB)
DPB2.2 ---------------------------LV------------------E-------------E--------------------- (QBL)
DPB8 ----------------------------V------------------DE--------- --E------V-------DEAV--- (Plaz) O
DPB5 _ ___________--------------LV------------------E----------------------------DEAV--- (HAS)
DPB7 --------------------------------------------------------------------V-------DEAV--- (Raji)
DPB3 VY-L------------------------V------------------DED-------L----------V-------DEAV--- (SLE)
DPB6 VY-L------------------------V------------------DED-------L---E--------------DEAV--- (JMOS)
DPBll VY-L---------------------Q-Y-----------------------------L---R--------------DEAV--- (CRK)
DPB13 VY-L-----------------------Y---------------------------------E------I-------DEAV--- (BH)
DPBl VY-------------------------Y----------------------------------------V-------DEAV--- (LUY)
DPB10 VH-L------------------------V------------------DE------------E------V-------DEAV--- (BM21)
DPB9 VH-L------------------------V------------------DED-----------E------V-------DEAV--- (TOK) ~~~
DPB12 --VH-L-----G---------S--------V------------------DED-----------E------V--R----DEAV----- (AKIBA) C~
DPB14 VH-L------------------------V------------------DED-------L----------V-------DEAV--- (8268) C~
DPB15 VY-----------------------Q-Y-----------------------------L---R--------------V------ (PLH) CS~DPB16 ----------------------------V------------------DE--------,---E--------------DEAV--- (JRA)
DPB17 VH-L------------------------V------------------DED-----------E--------------DEAV--- (JRAB)
DPB18 VY________ ____-------------V------------------DE---------------------------V------ (JCA) ~
DPBl9 ----------------------------V------------------E------------DE------I-------DEAV--- (CB68)
DPB20 VH-L------------------------V------------------DED-------L------------------DEAV---
SXB -SVY-E------------VVDGL------YVH--A----L--M------IG--F-----FM-R---EV-KV---K---ME-LIR--- (Priess)
1339206
To detect and distinguish between DP alleles in a practicable and economic fashion,
however, one must know the nuc]eotide sequence of the alleles. Portions of the nucleotide
sequences of various DPalpha and DPbeta alleles are shown below in Table II; thesequences are identified as above in Table I. The illustrative primers of the invention
enable production of DNA from which the sequence of codons 8 to 90 can be deterrnined.
The location of allele sequences that are the preferred target sequences for hybridization
with the various probes of the invention are designated as ¦ -A- ¦, ¦ -B- ¦, ¦ -C~ F- ¦ .
I -D- I 1 -E- I and I -F- I .
r~
TABLE II
+10 +15 +20 +25
AsnTyrLeuPheGlnGlyArgGlnGluCysTyrAlaPheAsnGlyThrGlnArgPheLeuGluArgTyr
DPB4.1: AGAATTACCTTTTCCAGGGACGGCAGGAATGCTACGCGTTTAATGGGACACAGCGCTTCCTGGAGAGATACDPB4.2: ---------------------------------------------------------------DPB2.1: -------------------------------------------~---~~~~~~ ~
DPB2.2: -----------------------------------~~--~~~~~~ ~
DPB8: ---------------------------------------------------------------
DPB5: ____________________________________________ ______________ ___
DPB7: ---------------------------------------------------------------
DPB3: G-G-A----TT----------------------------------------------------
DPB6: G-G-A----TT----------------------------------------------------
DPB11: G-G-A----TT----------------------------------------------------
DPB12: --------G-GCA----TT-----------------GC-----------------------A-----T---
DPB13: G-G-A----TT----------------------------------------------------
DPB1: G-G-A----------------------------------------------------------
DPB9: G-GCA----TT----------------------------------------------------
DPB10: G-GCA----TT----------------------------------------------------
DPB14: G-GCA----TT----------------------------------------------------
DPB15: G-G-A----------------------------------------------------------
DPB16: --------------------------~---~~~-~~~~~~~~~~~~~~~~~~
DPB17: G-GCA----TT---------------------------------------------------- C~
DPB18: G-G-A----------------------------------------------------------
DPB19: --------------------------------------------------------------- ~
DPB20: G-GCA----TT---------------------------------------------------- O
I-----A-----I
%
TABLE II
+30 +35 +40 +45 +50
IleTyrAsnArgGluGluPheAlaArgPheAspSerAspValGlyGluPheArgAlaValThrGluLeu
DPB4.1: ATCTACAACCGGGAGGAGTTCGCGCGCTTCGACAGCGACGTGGGGGAGTTCCGGGCGGTGACGGAGCTG
DPB4.2: ------------__________T----------------------------------------------
DPB2.1: -----------___________T-----------------------------_________________
DPB2.2: ------------------C---T----------------------------------------------
DPB8: ----------------------T----------------------------------------------
DPB5: ------------------C---T----------------------------------------------
DPB7: -------------------~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~
DPB3: ----------____________T--------------------------____ _______________
DPB6: ----------------------T----------------------------------------------
DPB11: ------------C------A------------------------A------------------------
DPB12: -----------___________T-----------------------------___ ____________
DPB13: -------------------A------------------------A------------------------
DPB1: -------------------A-------------------------------------------------
DPB9: ----------------------T------------------------------------____-_____
DPB10: ----------------------T----------------------------------------------
DPB14: ----------------------T-----------------------_________ _____________
DPB15: ------------C------A------------------------A------------------------ ~'
DPB16: ----------------------T-------------------___________________________ C~
DPB17: ----------------------T----------------------------------------------
DPB18: ----------------------T----------------------------------------------
DPB19: ----------------------T---------------------------------------------- O
DPB20: ----------------------T----------------------------------------------
I-----B-----I
~7
~ i
TABLE II
+55 +60 +65 +70
GlyArgProAlaAlaGluTyrTrpAsnSerGlnLysAspIleLeuGluGluLysArgAlaValPro
DPB4.1: GGGCGGCCTGCTGCGGAGTACTGGAACAGCCAGAAGGACATCCTGGAGGAGAAGCGGGCAGTGCCG
DPB4.2: -- ______-A--A--------------------------------____________________
DPB2.1: _______ _-A--A--------------------________________-G------_-______
DPB2.2: _________-AG---------------------------------------G--------------
DPB8: __ _____-A--A------------------____ _____________-G------_-______
DPB5: _________-AG----------------------------------__--_____ __ _______
DPB7: ------------------------------------------------------------------
DPB3: ----------A--A---C---------------------C--------------------------
DPB6: ----------A_-A---C---------------------C-----------G-------------- r
DPB11: ---------------------------------------C------------G-------------
DPB12: ----------A--A---C---------------------------------G--------------
DPB13: ---------------------------------------------------G--------------
DPB1: --------------------------~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ ~
DPB9: ----------A--A---C---------------------------------G--------------
DPB10: -- ______-A--A-------------------_-_ ___________ _-G------_____ __
DPB14: ----------A--A---C---------------------C-------------------------- ~ '
DPB15: ---------------------------------------C------------G------------- C~
DPB16: ----------A--A-------------------------------------G-------------- C~
DPB17: ----------A--A---C---------------------------------G--------------
DPB18: ----------A--A---------------------------------------------------- ~
DPB19: ----------AG---------------------------------------G-------------- ~
DPB20: ----------A--A---C---------------------C--------------------------
I---C----I l------D-------I
TABLE II
+75 +80 +85 +90
AspArgMetCysArgHisAsnTyrGluLeuGlyGlyProMetThrLeuGlnArgArg
DPB4.1: GACAGGATGTGCAGACACAACTACGAGCTGGGCGGGCCCATGACCCTGCAGCGCCGAG
DPB2.1: ---------------------------------------------------
PB2.2: ------------------
DPB8: ------G-A----------------------A--A-G--G-----------
DPB5: -------------------------------A--A-G--G-----------
DPB7: ------G-A----------------------A--A-G--G-----------
DPB3: ------G-A----------------------A--A-G--G-----------
DPB6: -------------------------------A--A-G--G-----------
DPB11: -------------------------------A--A-G--G-----------
DPB12: ------G-A-------G--------------A--A-G--G------------------
DPB13: --------A----------------------A--A-G--G-----------
DPB1: ------G-A----------------------A--A-G--G-----------
DPB9: ------G-A----------------------A--A-G--G-----------
DPB10: ------G-A----------------------A--A-G--G-----------
DPB14: ------G-A----------------------A--A-G--G-----------
DPB15: --------------________________-T---------______ ___
DPB16: -------------------------------A--A-G--G----------- C~
DPB17: -------------------------------A--A-G--G----------- r~
DPB18: -------------------------------T------------------- ~
DPB19: --------A----------------------A--A-G--G----------- ~
DPB20: -------------------------------A--A-G--G-----------
I-E-I l----F----I
I 133920~
26
The DNA sequences provided above are an important aspect of the present
invention. Although only one strand of the sequence is shown, those of skill in the art
recognize that the other strand of the sequence can be inferred from the information
depicted above. This information enables the construction of the probes of the invention.
Many illustrative probes of the invention are shown in the Examples below. However,
suitable SSO probes for hybridization analysis of the DPbeta alleles will comprise (or be
complem.ont~ry to) certain polymorphic sequences. Six sets of illustrative probes of the
invention are depicted below; each set is designed to distinguish between the
polymorphisms in a specific segment of the second exon of the HLA ~DPbeta gene. The
designation of the segments is as described above. The polymorphic residues encoded
within the allelic variant in the segment to which a probe hybridizes is shown in one letter
amino acid code (the dash means that prototypic residues are present in those positions) to
the left of the probe sequence. The probes span the regions encoding the polymorphic
amino acid residues and are shown as having a length of about 18 nucleotides. Those
sequences in the probe that encode the polymorphic amino acid residues, and thus must be
included within a probe for detecting the alleles that encode the de.sign~tçd segment, are
between the slash marks in the sequence. The DP alleles with which the probe will
hybridize are shown to the rjght of the probe.
13~9206
HLA DPbeta SSO Probes
Amino Acid Probe Sequence DPbeta Alleles
SegmentA:LF-G:TAC\CTTTTCCAGGG\ACGG 2.1, 2.2, 4.1, 4.2, 5, 7, 8,
19
VY-L: TAC\GTGTACCAGTT~CGG 3, 6, 11, 13
VY-G: TAC\GTGTACCAGGG\~CGG 1,15, 18
VH-L: TAC\GTGCACCAGTT\~CGG 9, 10, 12, 14, 17, 20
SegmentB:E-FA:CGG\GAGGAGTTCGC\GCGC 4.1, 7
E-FV: CGG\GAGGAGTTCGT\GCGC 2.1, 3, 4.2, 6,- 8, 9, 10,
12, 14, 16, 17, 18, 19, 20
E-LV: CGG\GAGGAGCTCGT\GCGC 2.2, 5
1 0 Q-YA: CGG\CAGGAGTACGC\GCGC 1 1 ,1 5
E-YA: CGG\GAGGAGTACGC\GCGC 1, 13
E-FV: CGG\GAGGAATTCGT\GCGC 10
Segment C:AAE:CCTG\CTGCGGAG\TACTGG 1, 4.1, 7, 11, 13, 15
DEE: CCTG\ATGAGGAG\TACTGG 2.1, 4.2, 8, 10, 16
EAE: CCTG\AGGCGGAG\TACTGG 2.2, 5, 19
DED: CCTG\ATGAGGAC\TACTGG 3, 6, 9, 12, 14, 17, 20
SegmentD:I---K:GAC\ATCCTGGAGGAGAA\G 1, 4.1, 4.2, 5, 7, 18
I---E: GAC\ATCCTGGAGGAGGA\G 2.1, 2.2, 8, 9, 10, 12, 13,
16, 17
L---K: GAC\CTCCTGGAGGAGAA\G 3, 14, 20
L---E: GAC\CTCCTGGAGGAGGA\G 6
L---R: GAC\CTCCTGGAGGAGAG\G 11, 15
SegmentE:M: GACAGG\ATG\TGCAGACAC 2.1, 2.2, 4.1, 4.2, 5, 6,
11, 15, 16, 17, 18, 20
V: GACAGG\GTA\TGCAGACAC 1, 3, 7, 8, 9, 10, 12, 14
2s SegmentF:GGPM:CTGG\GCGGGCCCA\TGACC 2.1, 2.2, 4.1, 4.2
DEAV: CTGG\ACGAGGCCG\TGACC 1, 3, 5, 6, 7, 8, 9, 10, 11
VGPM: CTGG\TCGGGCCCA\TGACC 1 5,1 8
Because the probes of the invention are single stranded for use in hybridization, it is
important to note that merely because a probe is designed to hybridize with, for example,
the codi~g s~and, does not mean that an equally useful probe could not be designed that
would hybridize to the complementary sequence present on the noncoding strand.
The seq~ence information provided above also relates to other illlpc,~ t aspects of
the invention. The preferred primers of the invention are designed to amplify many
different DP alleles. In many instances, as demonstrated in the Examples below, such
primers are very useful. However, those of ski~l in the art reco~nize that the DNA
13~920~
28
sequence information provided above can also be used to design primers that will enable
allele specific amplification. Such allele-specific primers will only amplify a single allele or
a certain subset of known alleles. For example, the "DEAV" probes of the invention can be
used as one primer of a primer pair to provide for allele-specific amplification of "DEAV"
DPbeta alleles.
The present invention also relates to kits that comprise a multicontainer unit
comprising essential components for practicing the present method. For example, the kit
can contain primers for PCR, as such primers are necessary in the preferred embodiment
of the invention. These primers will amplify at least the DPbet~ gene, and, whena~p~ ,iate, for example in forensic analysis, primers can be included that also amplify the
DPalpha gene. The kit must also contain SSO probes for at least the DPbeta gene, and,
when appropriate, for the DPalph~ gene as well. In some cases, the SSO probes may be
fixed to an appropriate support membrane which is useful for the hybridization analysis.
Other optional components that may be contained in containers within the kit include, for
example, an agent to catalyze the synthesis of primer extension products, the substrate
nucleotides, means used to label (for example, an avidin-enzyme conjugate and enzyme
substrate and chromogen if the label is biotin), and the ~,t)plo~liate buffers for PCR or
hybridization reactions. In addition to the above components, the kit can also contain
instructions for carrying out the present method.
A number of examples of the present invention, which are provided only for
illustrative purposes and not to limit the scope of the invention, are presented below.
Numerous embodiments of the invention within the scope of the claims that follow the
examples will be apparent to those of ordinary skill in the art from reading the foregoing
text and following examples. In the following examples, certain techniques were standard,
unless specifically indicated otherwise. Such techniques include PLT, which was
performed essentially as described by Shaw et ah, 1980, J. Exp. Med. 152:565 580.
Generally, for Iymphocyte priming, responder and stimulator cells were thawed, washed,
and resuspended in RPMI- 1640 medium supplemented with glutamine and antibiotics(complete media). Responding cells were mixed with irradiated stim~ tor cells in a ratio of
2:1, and the cell mixture was incubated for ten days at 37 degrees C. The primed irradiated
stimulator cells were co-cultured with irradiated stim~ tor cells in complete media. After
48 hours, 3H-thymidine was added to the culture. The cells were harvested 18 hours later,
and tritium incorporation was evaluated by coun~ing beta-emission.
1~39206
29
DNA sequence analysis was per~ormed as described by Maniatis et ah, Molecular
Cloning: A Laboratory Manual (New York, Cold Spring H~rbor Laboratory, 1982).
Generally, the sequences to be analyzed were cloned into an M13 cloning vector and
analyzed by either the Maxam-Gilbert technique or by the dideoxy chain termination
technique. Synthetic oligonucleotides, both primers and probes, were synthesized using
colll,J,elcially available instruments and techniques well known in the art.
Example 1
Analysis of the DNA Sequences of HLA DP Alleles
The DNA sequences of the variable second exons of a variety of alleles of the
DPalpha gene and of the DPbeta gene were determined. The DNA samples used were
chosen to represent as wide a spectrum of PLT defined DP alleles as possible. DNA was
extracted from cell lines homozygous for the standard six DPw types, from cells exhibiting
unusual typing reactions, and from cells exhibiting DP blank reactions. DNA extraction
was by standard techniques, as described by Maniatis et al., in Molecular Cloning: _
Laboratory Manual. The variable second exons of the DPalpha and the DPbeta genes were
arnplified by the PCR method, as described. The amplified DNA sequences were cloned
into an Ml3 derived vector, and the DNA sequences were determined by the chain
termination method. The cell lines used, their DR serotypes, their PLT defined DPw types,
and the DNA defined alleles they were found to contain are listed in tabular form below
(blank spaces indicate data not determined).
1339205
Gell DR DPw DPB DPA
Llne Tvpe Tvpe Alleles Alleles
CRK 7 1* 1, 11
RSO101 w6 1, 3 1, 3
BMG 4 1, 4 4.1
QBL 3 2 2.2
WJR 2 2 2.1
PJM 3 2, 3 2.1, 3
RMD 5,w13 2,4 2.1,4.1
.~MOS 4,5 2,6 2.1,6
SLE w13 3 3
COX 3 3 3
(~PR w8, wl0 3, 4 3, 4.1
JAH 4 3, 4 3, 4.1
MRI 1, 2 3, 5 3, 5
HHK w6 4 4.1
LBI 3,7 4,MAS 1,4,4.2
APD w6 4* 2, 4
LUY w8 1, 4 1, 4.1 1, 2
WDV w6 2, 4 2.1, 4.1
SMF 2, w12 4, 5 4.1, 5
BCR 4, w6 4, 6 4.1, 6
GTER w8, ~ 5 5
HAS 4 5 5, 19
DKY 9 5 2.1
BIN4() 4 3,6 3,6
LG2 1 ? 4.1
PIAZ 2 . 7 8
TOK 2 9 2
BM21 wl 1 10
VLA 2, 3 11
GBA 1, 7 11
CDI 3, 4 3, 6 10
CD2 7, (5) 2, 4 4.2, 10
CD8 3, 7 4 4.1, 4.2
CDIl 2 2.1, 4.2
DP "MAS" is provisional designation for a newly defined DP specificity related to the
DPB4.2 allele. "CD" cell lines actually are samples from CD patients.
* - refers to cells with unusual DPw phenotypes.
1339~0~
31
As shown from the foregoing, the DNA analysis of the HLA DP subtypes shows
that specific sequences correlate with the known PLT-defined DPw typings, in~lica~ing that
the polymorphic epitopes recognized by the primed T cells are on the DPbeta chain. For
some DP types, e.g., DPw2 and DPw4, sequence analysis has revealed subtype variants.
The variants for DPw2 have been designated DPB2. 1 and DPB2.2; cells typed for PLT as
DPw4 "new" (e.g., LB1) or DPw4* (e.g., APD) contain the more rare DPB4.2 subtype.
The DPB4.2 subtype is more related by sequence to the DPB2. 1 allele than to the DPB4. 1
allele. Individual CDl l is PLT typed as DPw2, but contains the closely related DPB2. 1
and DPB4.2 alleles.
The results above also show that unique DPbeta sequences correspond to the
DPw l, DPw3, DPwS, and DPw6 specificities, and these alleles have been designated to
reflect this correlation. A few exceptions are, however, that cell line DKY has been typed
as DPw5, but contains the DPB~. I allele, and that individual CD2 is PLT typed as DPw2,
but contains the DPB4.2 and DPBI0 alleles.
Example 2
PCR Ampliflcation of the DPalpha and DPbeta Genes
The DPalpha and DPbeta genes of some of the cells described in Example l were
amplified by PCR. The primers used, which were synthetic, are shown below. In this
depiction, the left side primers, GH98 and DB01, are from the upper strand, and direct
DNA polymerase to extend rightward. The right side primers, GH99 and DB03, are from
the lower strand, and direct synthesis leftward. The areas of the genes to which the
primers bind, and which will act as templates for primer extension are also shown. Lower
case letters indica~e bases in the primer that are not complementary to the target genomic
DNA ~shown in the opposite strand). These changes in the primers incorporate restriction
enzyme sites (BamHl or Pstl) at the ends of the amplifled DNA and facilitate cloning of the
amplified DNA. The oligonucleotide primers GH98 and GH99, which are used for theamplification of the second exon of DPalpha, amplify a 243 bp segment. The first two bp
of the PCR product are from the intervening sequence which flanks the exon. The
oligonucleotide primers DB01 and DB03 amplify a 294 bp segment of the second exon of
DPbeta. The left 13 bp and the right 17 bp of the product are from the intervening
sequence.
DP~l~h~ Prl~rs
GH98 > Phe Val Gln Thr Hls Arg Pro Thr ................... Leu Asn Asn Asn Le~ Asn Thr Leu Ile
cGcGGAtccTGTGTcAAcTTATGccGcG TTT GTA CAG ACG CAT AGA CCA ACA ........ TTG AAC AAC AAC TTG M T ACC TTG ATC CAGCGTTCCAACCACACTCAGGCCAC
TCGCCTGGTACACAGTTGAATACGGCGC AAA CAT GTC TGC GTA TCT GGT TGT ........ AAC TTG TTt, TTG M C TTA TGG AAC TAG GTCGCAAGGTTGGTGTGAcgtCGGTc
<~ GH99
DPbeta Prll-rs
------- DB01 --~ -> Leu Phe Gln Gly Arg Gln Glu Cys Tyr .......... Glu Leu Gly G1y ~to Met Thr Leu Gln
CagggatCCGCAGAGAATTAC CTT TTC CAG GGA CGG CAG GAA TGC TAC ........ GAG CTG GGC GGG CCC ATG ACC CTG CAG CGCCGAGGTGAGTGAGGGCTTTGG
GGGGAGGGGCGTCTCTTAATG GAA AAG GTC CCT GCC GTC CTT ACG ATG ........ CTC GAC CCG CCC GGG rAC TGG GAC GTC GCGGCTCCACTCACTgaCGtcctg
c-------- DD03
1~92~
33
Hybridization of the primers and the synthesis of the elongated primer containing products
were essentially as described in EP 258,017 and in the protocols provided by themanufacturer, PECI, of the Thermal Cycler used to perform the PCR. Amplification was
for 28 cycles; however, more, i.e., 35, cycles can on occassion yield better results.
Two other illustrative primers of the invention for amplifying the second exon of
s DPbeta alleles are designated UG l 9 and UG2 l . The sequences of these primers and the
other illustrative DPbeta primers of the invention are shown below.
DPbeta Primers
Designation Sequence Side
DBOI CAGGGATCCGCAGAGAATTAC Left
l 0 DB03 GTCCTGCAGTCACTCACCTCGGCG Right
UG l 9 GCTGCAGGAGAGTGGCGCCI CCGCTCAT Left
UG2 I CGGATCCGGCCCAAAGCCCI CACTC Right
Example 3
SSO Probej for Hybridization Analysis of DPbeta Alleles
Illustrative probes of the invention referred to throughout the remainder of theExamples are described below in tabular form. In the Table, the probe designation, probe
sequence, polymorphic amino acid sequence encoded in the region of the allele to which the
probe hybridizes, segment designation, and hybridization and wash conditions are shown.
Probes are shown as having a 32p or "X" label, where X represents HRP, as discussed in
the Examples below. Where a probe sequence is indicated by an X followed by a probe
designation, the sequence of the probe is identical to that of the probe designated after the
X, except that the 32p label has been replaced by an HRP label.
Those of skill in the art recognize that depending on the type of label used,
hybridization and wash conditions will differ. Although, in a plefellcd embodiment, the
probes will be labeled nonisotopically (e.g., with HRP), some of the probes have been
used with isotopic (e.g., 32p) labels. Consequently, hybridization and wash conditions for
32P-labeled and HRP-labeled probes are shown, where such conditions have been
empirically determined (see Bugawan et ah, 1988, l. Immunol. 141(l2):4024-4030). In
the Table, the conditions referred to assume a hybridization solution composed of 5 x
I~enhardt's solution, 0.5~G SDS, and the indicated amounts (i.e., O.l x, 3 x, 5 x) of SSPE.
Five x Denhardt's solution contains ().5 g Ficoll, 0.5 g polyvinylpy~Tolidone, 0.5 g BSA
(Pentax Fraction V) per 500 ml. The ~ash so~ution conta~ns 0.1 x SSPE and 0.1% SDS
1339206
~4
(for HRP-labeled probes, 0.1% Triton X- 100 was used in place of SDS); the wash step is
carried out at the indicated temperature for ten minutes. As described in Example 11,
however, the use of tetramethyl ammonium chloride, or similar salts, can be used to allow
for more uniform hybridization and wash conditions, a preferred condition when a number
of probes are used in a panel to determine the types of DP alleles in a sample.
1339206
SSOs for HLA DPbeta Typing
Probe Arnino Acid Probe Sequence Re~ion Hybridiz'n/Wash
Designation Sequence Conditions
DB10 LFOG 32P-GAATTACCTTTTCCAGGGA A 5 x ~? 50~/42~ H2O bath
DB27 LFOG X-DB 10 A 5 x ( ? 50~ /42~ air
DB 11 VY(~L 32P-ATTACGTGTACCAGTTACG A 3 x ( ) 55~/42~ H2O bath
DB28 VY~L X-DB 11 A 3 x ( ? 55~/42~ H2O bath
DB58 VYQL X-ATTACGTGTACCAGTTA A 3-5 x@ 42~/42~ air
DB23 WQL X-CGTAACTGGTACACGTAAT A 5 x (? 50~/42~ H2O bath
DB36 VYQL X- DB23 A 5 x ( ? 50~/42~ air
DB12 VYQG 32P-CGTCCCTGGTACACGTAAT A 5 x ( ? 50~/42~ H2O bath
DB29 VYCG X-DB 12 A 5 x ( ? 50~/42~ H2O bath
DB22 VHQL 32P-ATTACGTGCACCAGTTACG A 3 x ( ? 55~/42~ H2O bath
DB35 VHC~L X-DB22 A 3 x( ? 50~/42~ H20 bath
DB13 AAE 32P-CCTGCTGCGGAGTACTG C 3 x ( ? 55~/42~ H20 bath
DB30 AAE X-DB 13 C 3 x ( ? 50~/42~ H2O bath
DB14 DEE 32p-cAGTAcTccTcATcAGG C 5 x ( ? 42~/42~ H20 bath
DB31 DEE X-DB 14 C 5 x ( ) 42~/42~ H2O bath
DB16 EAE 32p-cAGTAcTccGccTcAGG C 5 x ( ? 42~/42~ H20 bath
DB32 EAE X-DB 16 C 3-5 x @ 42~/42~ H2O
bath
DB59 EAE X-CCTGAGGCGGAGTACTG C 5 x (? 50~/42~ H2O bath
DB17 DED 32P-CCTGATGAGGACTACTG C 5 x ( ? 50~/42~ H2O bath
DB33 DED X-DB 17 C S x ( ? 50~/42~ air
DB18 I-K 32P-GACATCCTGGAGGAGAAGCD O.lx @ 55~/42~ H2O
bath
DB34 I-K X-DB18 D 3 x ~? 55~/42~ H2O bath
DBl9 I-E 32P-GCTCCTCCTCCAGGATGTC D 5 x ( ? 50~/42~ H2O bath
DB37 I-E X-DB 19 D S x ( ? 55~/42~ H2O bath
DB20 L-K 3~P-GACCTCCTGGAGGAGAAGC D 3 x ( ? 55~/42~ H2O bath
DB38 L-K X-DB20 D 5 x( ) 55~/42~ H2O bath
DB21 L-E 32P-GCTCCTCCTCCAGGAGGTC D 5x(?50~/42~H20bath
DB39 L-E X-DB21 5 x ( ? 50~/42~ air
DB62 L-E X-GACCTCCTGGAGGAGGAG D 3 x ( ? 50~/42~ H2O bath
DB63 L-R X-GACCTCCTGGAGGAGAGG D 3 x ( ? 50~/42~ H2O bath
DB25 GGPM 32P-CTGCAGGGTCATGGGCC-
CCCG F 5 x @ 50~/42~ H2O bath
DB40 GGPM X-DB25 F 3 x@ 50~/42~ H2O bath
DB26 DEAV 32P-CTGCAGGGTCACGGCCT-
CGTC F 5 x @ 50~/42~ H2O bath
DB41 DEAV X-DB26 F 3 x @ 50~/42~ H2O bath
With reference to the Table above, one should note that because DB28 cross-
hybridizes with DB32, superior results can be obtained using probes DB58 and DB59 in
place of DB28 and DB32, respective}y. ln addition, probe DB63 is preferred over DB39.
1339206
3~
Example 4
SSO Probes for Hybridization Analysis of DPalpha Alleles
Exarnples of suitable SSO probes for hybridization analysis of DPalpha alleles are
shown below. Two sets of probes are illustrated; each set is designed to distinguish
between the polymorphisms in a specific segment of the second exon of the HLA DPalpha
gene. ASOI and ASO2 bind to the DPAl allele at the region cont~ining the polymorphic
segrnents containing methionine (M) and ~lul~..,;t~e (O), respectively. ASO3 and ASO4
bind to the DPA2 allele to the region cont~inin~ the polymorphic se~ ,cnls which contain
glutarnine and arginine (R), respectively. ASO2 and ASO4 distinguish the polymorphic
- 10 segment containing glutamine from that containing arginine. The probes span the regions
enCo~1ing these polymorphic amino acid residues. Hybridization using these probes is
usually carried out in a solution containing 5 x SSPE, 5 x Denhardt's, and 0.5% SDS, for
at least I hour at 42~C. Also shown are the washing conditions for use with the probes.
HLA DPalpha SSO Probes
Probe Sequence Washing Conditions
ASO1AGATGAGATGl~CI'ATG 2 x SSPE, 0.1% SDS, 42~
ASO2Gl l'I'GGCCAAGCCTI~ 2 x SSPE, 0.1% SDS, 50~
ASO3AGATGAGCAGTTCTATG 2 x SSPE, 0.1% SDS, 50~
ASO4GlTlGGCCGAGCCTlTI 2 x SSPE, 0.1% SDS, 55~
Example 5
Analysis of Amplified DPbeta Sequences by Hybridization with SSO Probes
PCR amplified DPbeta sequences from 24 HTCs were analyzed in a dot blot format
with a panel (n=9) of 32P-labeled SSO probes, and the DP type was inferred from the
pattern of probe binding. The extraction of DNA from the cells was as ~es~ibed in
2s Example 1. The target regions of the cellular genome, i.e., the second exon of the DPbeta
genes, were amplified by the PCR t~chni~ue as described in Example 2, except that the
DNA was from tl7e ceUs listed in tabular form above.
The amplified DNAs were dot blotted onto a filter, a separate filter containing the
panel o~s~ s was }~ ~paled for analysis by hybridization with each SSO probe. To dot
blot the samples, five microliters of each amplified sample were diluted with 195
microliters of a solution containing 0.4 N NaOH and 25 mM EDTA and spotted onto 9
37 1339206
replicate Genatr~n 45 (Plasco) nylon filters by first wetting the filters with water, placing
them in a Bio-dot (Bio-Rad, Richmond, CA) apparatus for preparing dot blots, applying
the samples, and rinsing each well with 0.4 ml of 20 x SSPE (3.6 M NaCl, 2Q0 mM
NaH2PO4, and 20 mM EDTA). The filters were removed, rinsed in 2 x SSPE, and baked
for 30 minutes at 80~C in a vacuum oven.
The samples on the filters were hybridized with SSO probes of the invention.
Hybridization was with Q.25 to 0.5 pmoles of probe in 2 to 5 ml of hybridization solution.
Hybridization and wash conditions were as described in tabular form in Example 3. The
results of this DPbeta typing are shown below, which also shows the probe with which the
samples on the filter were hybridized and the encoded amino acid sequence detected by the
probe.
1339206
38
oo
x ~ ôo x oô î~ ~ x
D ~ ~ ~ ~ ~ ~ ~~ O _~ O
G _ ~ o ~ '-- . . . . . . . . . . .
~ ~D ~
~ + ' ' ' ' ' ' ' + + + ' ' ' ' ' ~ C,C o
~i 3 o ~
î ~ + ~ + ~ +
oo ,_ ~' . '' o
~. '' ~
D ~ + + + + , , I , , ~ ~ ~ I I I I + _ 3 v ~ ~
'J ~ _ C
~ a a~ ~ ~ ' ' + + + + + + ' ' ' ' ' + ' ' + + ' ' + + ' ' 3 ~ o
~ ~ .~.C~D ~
+,, .,,,,,, + +, + +, + + +,,,, ~ ~ f~ 2 c
a ~, + , , , ~ 9
+ t ~ ~ I I I t + + + I I ~; I I I I I 1 6 ~a C ~ C
o V V. ~ .' 3 ~ D
V ~ C
D D C S ~; ~ c
_ S ~ C~
O ~ 6 o
) ~ m m m ~ , m ~ 6 m ~ c 3 o ~ D
* * * * * C~
133920~
39
The determination of DPbeta types of the cells based upon the hybridization
analysis with the SSO probes has been discussed above. To determine the DPbeta type of
a sample, the binding of the probes to the sample was ex~mined The alleles present were
inferred from the pattern of probe binding. For example, sample 1 formed hybrids with
SSO probes DB 11, DB 17, and DB20. The amino acids encoded by DB 11, DB 17, and
DB20 are VYQL, DED, and LEEK, respectively. An e~min~tion of segments A, C, and
D of the DPbeta allelic amino acid sequences shows that the sequence VYQL is present in
DPB6, DPB 11, and DPB 13; the sequence DED is present in DPB 17, DPB 14, -DPB 12,
DPB9, DPB6, and DPB3; the sequence LEEK (L K) is present in DPB18, DPB14,
DPB4.1, DPB4.2, DPBS, DPB7, DPB3, and DPBI. The only allele which contains the
three sequences which hybridize with the probe is DPB3. Thus, the DP type of sample 1
based on SSO typing is DPB3.
The DPB types of the other samples were inferred by the same type of analysis, and
the determined types are depicted above. In the depiction, the asterisk (*) indicates cells
where the DPbeta genotype was also determined by sequence analysis. The DPw type of
the cell ]ines COX (formerly wl ~w3) and BM21 (formerly wl~blank) have recently
been changed to the type indicated. The symbol + refers to a weak signal obtained with a
probe. In some cases (BM21 and TOK), this weak signal reflects the presence of an
additional polymorphic sequence in region A encoding the amino acid residues VHYL,
which cross-hybridizes to the DB 11 probe. The sequence can be more conveniently typed
with region A probe DB~2. For other cell lines (BM92), there is apparently a background
cross-hybridization of the DBI I probe to the sequences recognized by the DB10 probe. In
sirnilar fashion, cross-hybridization signals can occur with the DB 19 probe on sequences
complementary to the DB 18 probe.
The panel of SSO used in this Example detects variation at only 3 of the S
polymorphic regions and does not detect all allelic variants at these 3 regions. The typing
system using the procedure of this example is simple and unequivocal for HTCs, but given
the patchwork pattern of polymolphism, can occasionally give rise to ambiguous typing for
heterozygous individuals if the hybridization pattern of the various probes can be
interpreted as more than a unique pair of alleles. This ambiguity arises from the many
different combinations of the DPbeta sequence variants that constitute the different DPbeta
alleles. However, with the use of the additional SSO probes provided by the invention,
1339206
which additional probes span the remaining polymorphic regions, unarnbiguous typings
can be ob~ained for heterozygous individuals.
Example 6
Hl_A DP Typing of Coeliac Disease Patients
by DNA Sequence Analysis of PCR Amplifled Target Regions
The cells of four patients with coeliac disease (CD) were PLT-typed, and the DNAsequences of the second exon of the DPbeta determined as described in Example 1. The
CD diagnosis was based on clinical symptomology.
The results of the analysis obtained by DNA sequencing of the DPbeta (second
exon) of the CD eells has been discussed above. From these results, one observes that,
when CD cells are compared to non-CD cells, there is an apparent increase in the frequency
of the DPB4.2 allele~ In addition, the DPBI0 allele sequence is present in two independent
CD patients yet observed in only one of the 30 non-CD cell lines.
Example 7
HLA DP Typing of Coeliac Disease Patients
by SSO Probe Hybridization Analysis
The cells of l9 patients with CD and of 43 non-CD controls were analyzed for HLADP type by SSO probe hybridization analysis. The diagnosis of CD was based on clinical
symptomology; the CD patients as well as control individuals were all from Italy. DNA
extraction was as described in Example 1. PCR amplification of the samples was as
described in Example 2. Analysis of the amplified sequences was as described in
Example 4.
The results of the analysis showed that there was a significant increase in the
DPB4.2 allele in CD patients as compared to non-CD controls; this allele was present in 12
of 19 CD patients, but only 3 of 43 control patients. The DPB4.2 and DPB3 alleles were
present in 17 of 19 CD patients and in 15 of 43 controls. The genotype DPB4.1/4.2 was
present in 10 of 19 CD patients and in only 1 of 43 controls.
Example 8
HLA DP ~yping ~f Forensic Samples
Samples which con~ain genomic nucleic acids of the suspect individual are
obtained. The target regions of the genome, i.e., the regions containing the second exon of
1339206
41
the DPalpha and DPbeta genes, are amplified by the PCR technique, as described in
Example 2, except that the nucleic acids from the suspect replace those of the cells in the
Example, and except that the amplified samples contain a 32P-label. The amplified sample
is hybridized with the probes of the invention, which have been dot blotted onto the same
filter, and which have been immobilized on the filter by poly-dT tails. The hybridization
and washing conditions for the filter allow only perfectly matched hybrids to remain in
duplex state. The filters are examined to determine which probes form hybrids with the
labeled sample.
A sample which is to be compared with that obtained from the suspect is examinedfor DP type by the same procedure, i.e., by PCR amplification and hybridization with
immobilized SSO probes. The pattern of binding to the SSO probes of the sarnple from the
suspect and of the comparison sample is examined to determine the similarity or difference
in the hybridization patterns.
Example 9
HLA DP Typing Using SSO Probes
Labeled with Horseradish Peroxidase
A panel of cells from 39 individuals was typed for DPbeta alleles using SSO probes
for the second exon variants. The probes were labeled with horseradish peroxidase
(HRP), and hybrids detected in a dot blot format.
The cells analyzed were from fourteen IDDM patients, five DR3 control non-IDDM
patients, and nineteen HTCs which typed by PLT as DP blank. IDDM patients were
identified by clinical symptomology. DNA was isolated from the cells as described in
Example l. The target region, i.e., the second exon of the DPbeta alleles, was amplified
using the PCR technique in 200 microliters of reaction mixture that contained S0 mM Tris-
HC1, pH 8.3; 2.5 mM MgC12; l()0 micrograms/ml gelatin; 0.75 mM in each of the four
deoxynucleoside triphosphates; the primers DBOl and DB03; and Taq polymerase. The
ramping for amplification was the following: heating for 30 seconds to 94~C followed by
incubation at that temperature for 30 seconds; cooling for 1 minute to 55~C followed by
incubation at that temperature for 30 seconds; heating to 72~C for 30 seconds followed by
incubation a~ that temperature for 45 seconds. This cycling was repeated for 42 cycles.
A~ter amplification, the reaction mixtures wer~ sampled and monitored by gel
electrophoresis on gels containing 3% Nusieve, 1% Agarose to determine if all the DNA
amounts were comparable.
1339206
42
Filters containing the dot blotted samples were prepared by blotting 150
microliterstdot of denatured amplified DNA on Genatran nylon membranes and UV treating
the filters containing the samples for 5 minutes. The latter treatment is to fix the sample to
the membranes. The amplified DNAs were denatu~ed by treating 5 microliters of the PCR
reaction mixture in a total volume of 150 microliters cont~ining 0.4 N NaOH and 25 mM
EDTA. Eight replicate filters were prepared for hybridization with HRP labeled ASO
probes.
Prior to hybridization, the sample cont~ining filters were incubated for 15 rninutes
in pre-hybridization solution (1 x SSPE, S x Denhardt's solution, 1% Triton X-100)
without probe. In the pre-hybridization solution, Triton#~-100 was used in place of SDS.
Hybridization was in the same solution, which additionally contained 1 picomole probe/rnl.
Each filter was incubated for 40 minutes with 2.5 ml of hybridization solution containing
one of the HRP labeled probes. The probes used were DB27, DB28, DB29, DB30,
DB31, DB32, DB33, and DB35. The probes and hybridization conditions are listed in
tabular form in Example 3. After hybridization, the filters were washed as stated in
Example 3, under suitably stringent conditions, i.e., in 0.1 x SSPE, 0.1% Triton X-100
for 5 minutes at 42~C. The HRP-labeled SSO probes were prepared essenh~lly by the
methods disclosed in PCP publications 89/02931 and 89/02932.
These methods essentially involve derivatizing the nucleic acid probe using a linear
linking molecule comprising a hydrophilic polymer chain (e.g., polyoxyethylene) having a
phosphoramidite moiety at one end and a protected sulfhydryl moiety at the other end. The
phosphoramidite moiety couples to the nucleic acid probe by reactions well known in the
art (e.g., Beaucage et ah, 198~, Tetrahedron Lett. 22:1859-1862), while the deprotected
sulfhydryl group can forrn disulfide or other covalent bonds with the protein, e.g., HRP.
The HRP is conJugated to the 1inking molecule through an N-m~leimi(lo-6-aminocaproyl
group. The label is prepared by esterifying N-m~lçimido-6-aminocaproic acid with sodium
4-hydroxy-3-nitrobenzene sulfonate in the presence of one equivalent of
dicyclohexylcarbodiimide in dimethylforrnamide. After purification, the product is added
to phosphate buffer containing HRP at a weight ratio of 8:1 HRP to ester. The
oligonucleotide probe is synthesized in a DNA synthesizer, and the linking molecule having
the structure (C6Hs)3C~S-~CH2CH2O)4-P~ 2CH2CN)~N(i-PR~ ~tt~rh~ using
phosphoramidite synthesis conditions. The trityl group LS ~ loved, and the HRP
* Trade Mark
, .
f~
133~2~
43
derivative and probe derivative ~e mixed together and allowed to react tO form the labeled
probe. A biotin-labeled probe or primer may be prepared by similar methods.
Samples which contained hybridized probe were detected using a color developmentreaction, as described in Sheldon _ ah, 1986 Proc. Natl. Acad. Sci. USA 83:9085-9089,
5 which utilizes TMB/H202. The detection system is described in Example 10 below. The
HLA DP genotypes of the amplified DNA samples were readily apparent from the filters.
Example 10
HLA DPbeta Typin~ with HRP Labeled SSO Probes
lo A. PCR Amplification
DPB typing can utilize as many as or more than 14 SSO probes (sequence specific
oligonucleotides), so amplification is carried out on 0.5 to two micrograms of DNA, in 200
microliters of reaction volume, if DNA is not lirniting. Lower amounts of DNA, i.e., 100
ng, can be amplified, buI more cycles of amplification should be performed with such
15 sarnples, i.e., 45 cy~les.
The PCR reaction is started by mixing the following by vortexing for 1 to 2
seconds.
DNA 0.5 to 2 ~g
10 x Taq buffer 20 ~11
10() mM dNTPs 1.5 ~11
DPB primer [10 ~M UGI9 or DB01] 10 ,ul
DPB primer l lO ~M UG21 or DB03] 10 111
Taq polymerase 5 U/!ll 1.2 ~l
Glass-distilled H2O is added to achieve a final volume of 200 ~l. 10 X Taq salts are 500
mM KC1; 100 rnM Tris, pH 8.3; 15 rnM MgCI2; and 1 mg/ml gelatin. Negative controls
(i.e., no DNA) should be included in each PCR run. Typically, 30-35 cycles of
amplification in a Perkin-Elmer/Cetus Instruments DNA Therrnal Cycler are sufficient. The
cycles are designed to denature at 96~C for 30 seconds, and anneal and extend at 65~C for
30 seconds. If primer pair DB()l/DB()3 is used, annea~ing is at 55~C for 30 seconds and
extending is at 72~C for 30 seconds. An~tical ge~s can be ~sed to check PCRs and to
quantitate amount of DNA to be used for dot-~lots.
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44
B . Dot blot
Typically, 5 1l1 of the amplified DNA contain al)pr~ ately 200 ng, more than
enough for a single dot blot. Remember, however, that as many as or more than 14 dots
may be required, i.e., about 70 ~1 of the amplification reaction would then be used in
~ltp~ing the dot blots. For each S 111 of amplification reaction, 50 111 of 0.4 N NaOH and
25 mM EDTA are added to the DNA. Five minutes is sufficient to complete denaturation of
DNA. The Genatran membrane is first wetted in 2 x SSPE, and then the 150 ~1 of
denatured DNA are loaded into the dot blot apparatus. The m clllbl~ne is rinsed in 2 x
SSPE, and the DNA is fixed to the membrane with exposure to UV light for five minllte
i.e., by a 55 mJ/cm2 exposure in a Stratalinker 18001M UV light box, marketed byStratagene.
C. Hybridization
The ~"embl~ne is again wetted in 2 x SSPE, and about 5 ml of hybridization
solution per 8 x 12 cm membrane (size of dot blot app~L~Is) are added. About 1 to 1.5
picomoles of HRP probe are added per ml hybridization solution, and the probes are
allowed to hybridize for at least one hour. Hybridization solution is SSPE (as intlic~tetl
above), 5 x Denhardt's, and I % Triton X-100. The membranes are then washed with 0.1
x SSPE and 0.2% Triton X-100 for ten minutes. Otherwise, hybridization and wash
conditions were as described in Example 3. The probes used were DB27, DB29, DB30,
DB31, DB33, DB34, DB35, DB37, DB38, DB40, DB41, DB58, DB59, DB62, and
DB63.
D. Detection
The following steps for detection of probes are done at room tem~ldlu~; with
moderate shaking and just enough solution to completely cover the ~~le.l,b~ e, as is
described in Bugawan et ah, 1988, Bio/Technology 6:943-947. The detection is carried
out by a 5 minute incubation of the membrane with Buffer B, a 5 minute wash with Buffer
C, and 10 minutes incubation under light exclusion with Buffer C and TMB (48 ml of
Buffer C and 2.5 ml of 2 mg/ml TMB). Buffer B is 100 mM NaCl, 1 M urea, 5% Triton
X-100, and I % dextran sulfate. Buffer C is 100 mM sodium citrate, pH 5Ø TMB is 3,
3', 5, 5'-tetramelhylbenzidine. About 23 ,1l1 of 3% H2O2 are added to 50.5 ml of Buffer'
C/l'MB; the resulting solution is u~ed to de~elo~ d~e coEor on the n~m~r~nes (color comes
133920~
up within 1-5 minutes) under light-excluding conditions. The color development is
stopped by washing in H2O with a small amount of Buffer C. The wash is repeated twice
for 30 minutes. Pictures of the membrane are taken and the membrane is stored in Buffer
C under no light.
The methods described herein, as well as the SSO probes and primers, and kits
containing them, are useful for the accurate, relatively simple, and economic de~~ ination
of an individual's HLA DP genotype. Accurate DP typing will prove important in several
medical applications. For example, accurate HLA DP matching of donors and recipients
will be helpful in the prevention of allograft rejection and in the prevention of host versus
graft disease. Because certain HLA DP genotypes appear to be linked to certain
autoimmune diseases, including, for example, coeliac disease, myasthenia gravis, and
IDDM, HLA DP DNA typing is useful in an early diagnosis of the disease,~prior tomanifestation of full clinical symptoms.
Accurate HLA DP typing is useful in forensic medicine. For example, it provides
evidence as to whether a sample which contains genomic nucleic acids, for example, blood,
hair, or semen is derived from a suspect individual. It is also useful in detr~ g an
individual's paternity or maternity. The latter is of particular importance in analyzing
historical samples.
Example 1 I
Probe Hybridization in TMACL
When tetramethyl ammonium chloride (TMACL) is present in a hybridization
solution, probe discrimination is based on probe length and not on the G, C, A, or T
composition of the probe. Thus, by using TMACL in the hybridization solution, one can
hybridize and wash many different probes at a single temperature.
A suitable hybridization solution for this purpose contains 3 M TMACL; 0.5%
SDS; 10 mM Tris-HCl, pH = 7.5; and 0.1 mM EDTA. Hybridizations are carried out at
55~C for 30 to 60 minutes for l9-mer probes DB27, DB28, DB29, DB35, DB34, DB37,
DB38, and DB62; at 50~C for 17-mer probes DB30, DB31, DB33, and DB59; and at 60~C
for DB40 and DB41. The wash solution is 3 M TMACL; 50 mM Tris-HC1, pH = 8; and 2mM EDTA. The wash is carried out first at 37~C for 20 minutes, then at the higher
stringency temperature (the hybridization temperature) for 10 minutes. Detection of
hybridization is carried out as described in Example 10.
. I
133920~
46
Additional probes useful for this, or ~ny other, hybridization forrnat for purposes of
the present invention are shown below (X is HRP).
Probe Seqllence Length Amino Acid
Desi~nation Sequence
DB70 X-GAATTACCT~TCCAGGGAC 20-MER LFQG
DB92 X-GACCTCCTGGAGGAGGAGC 19-MER L-E
DBg3 X-GACCTCCTGGAGGAGAGGC 9-MER L-R
DB94 X-AGCTGGGCGGGCCCATGAC 19-MER GGPM
DB95 X-AGCTGGACGAGGCCGTGAC 19-MER DEAV
DB64 X-CTGGTCGGGCCCATGACC 18-MER VGPM
DB72 X-ACATCCTGGAGGAGAAGC 18-MER I-K
DB73 X-ACATCCTGGAGGAGGAGC 18-MER I-E
DB74 X-ACCTCCTGGAGGAGAAGC 18-MER L-K
DB71 X-TTACGTGTACCTGGGAC 17-MER VYQG
DB75 X-CCTGATGAGGAGTACTG 17-MER DEE
DB76 X-CTGGGCGGGCCCATG 15-MER GGPM
DB77 X-CTGGACGAGGCCGTG 15-MER DEAV
