U.S. patent application number 13/962182 was filed with the patent office on 2013-12-05 for means and methods for investigating nucleic acid sequences.
This patent application is currently assigned to Stichting Sanquin Bloedvoorziening. The applicant listed for this patent is Stichting Sanquin Bloedvoorziening. Invention is credited to Martin de Boer, Taco Willem Kuijpers.
Application Number | 20130323735 13/962182 |
Document ID | / |
Family ID | 40743878 |
Filed Date | 2013-12-05 |
United States Patent
Application |
20130323735 |
Kind Code |
A1 |
Kuijpers; Taco Willem ; et
al. |
December 5, 2013 |
MEANS AND METHODS FOR INVESTIGATING NUCLEIC ACID SEQUENCES
Abstract
The invention provides improved methods for investigating
nucleic acid sequences, wherein at least one additional probe is
used which is specific for a (pseudo)gene variant of a target
nucleic acid.
Inventors: |
Kuijpers; Taco Willem;
(Amstelveen, NL) ; de Boer; Martin; (Blokker,
NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Stichting Sanquin Bloedvoorziening |
Amsterdam |
|
NL |
|
|
Assignee: |
Stichting Sanquin
Bloedvoorziening
Amsterdam
NL
|
Family ID: |
40743878 |
Appl. No.: |
13/962182 |
Filed: |
August 8, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12998595 |
Jul 26, 2011 |
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PCT/NL2009/050669 |
Nov 5, 2009 |
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13962182 |
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Current U.S.
Class: |
435/6.11 ;
536/24.3 |
Current CPC
Class: |
C12Q 1/6858 20130101;
C12Q 1/6876 20130101; C12Q 2531/113 20130101; C12Q 1/6858 20130101;
C12Q 1/6883 20130101; C12Q 2600/156 20130101; C07H 21/04 20130101;
C12Q 2537/143 20130101; C12Q 2533/107 20130101; C12Q 2533/107
20130101; C12Q 2525/155 20130101; C12Q 1/6858 20130101; C12Q
2537/143 20130101 |
Class at
Publication: |
435/6.11 ;
536/24.3 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 5, 2008 |
NL |
PCT/NL08/50698 |
Claims
1-16. (canceled)
17. A probe or a probe set selected from the probes or probe sets
listed in FIG. 3A, 3B, 3C or 3D, preferably selected from the probe
sets of FIG. 3C or 3D.
18. Mixture of nucleic acids, wherein said nucleic acids comprise
at least two probes or probe sets according to claim 17.
19. A kit for detecting the presence of at least one target nucleic
acid sequence in a sample, comprising a probe or a probe set or a
mixture of nucleic acids according to claim 17.
20. A kit according to claim 19, wherein said at least one target
nucleic acid sequence comprises a nucleic acid sequence present in
a KIR locus.
21. A kit according to claim 19, further comprising a PCR primer
set comprising at least 70%, preferably at least 80%, more
preferably at least 85%, more preferably at least 90%, most
preferably at least 95% sequence identity to nucleic acid sequences
5'-GGGTTCCCTAAGGGTTGGA and TCTAGATTGGATCTTGCTGGCAC-3' or the
complements thereof.
22-32. (canceled)
Description
[0001] The invention relates to the fields of biology, molecular
biology, biotechnology and medicine.
[0002] Nucleic acid sequences are investigated in a wide variety of
applications. For instance, for diagnosis of infection with a
pathogen, a sample of an individual is often screened for the
presence of pathogen nucleic acid. Furthermore, nucleic acid
sequence investigation is often performed for the diagnosis of
genetic disorders, such as for instance Prader-Willi syndrome,
Angelman syndrome and Duchenne muscular dystrophy. Widely used
methods for detection of deletions or duplications of chromosomal
sequences are quantitative multiplex PCR and quantitative Southern
blotting. Drawbacks of these methods are that they are
time-consuming and that results are difficult to interpret.
[0003] One particularly suitable technique for investigation of
nucleic acid sequences is multiplex ligation dependent probe
amplification (MLPA). This technique is based on hybridisation of
probes to target nucleic acids, where after probes are amplified.
In currently used MLPA assays, each MLPA probe set consists of two
half probes. These two half probes contain a target-specific
sequence and a primer binding site sequence to which a nucleic acid
amplification primer (preferably a PCR primer) can bind. One half
probe is typically shorter in length then the other. The other half
probe is longer due to a non-hybridizing stuffer sequence. The
stuffer sequence of each probe set is unique in length, resulting
in different lengths of amplification products (typically between
130 and 480 base pairs) that can be separated by electrophoresis.
In an MLPA assay, typically a plurality of probe sets is used. The
two half probes of each probe set are typically added to denatured
sample nucleic acid and hybridized immediately adjacent to each
other on their target sequence. Subsequently, the resulting nucleic
acid is subjected to a ligation reaction. Usually a ligase is used
which ligates only half probes that are perfectly matched with
their target sequence (such as for instance the thermostable
Ligase-65). A mismatch of a half probe at the ligation site
prevents ligation and amplification. Thereby no amplification
products of the probe will be detected. This allows MLPA to
discriminate sequences that only differ in a single nucleotide.
Sequences from pseudogenes or related genes can therefore be
distinguished. Ligated half probes (which are also referred to as
"ligated probes") are amplified, preferably by PCR, using primers
capable of specifically binding the primer binding site sequences
of the probes. The amplification products of each ligated probe are
separated and analyzed, for instance by electrophoresis.
Preferably, amplification products are represented graphically by
separate peaks. Each peak is the product of an amplified MLPA
ligated probe and a relative difference in peak intensity (height
or surface) between a control sample and a sample of interest
indicates copy number variation. FIG. 1A schematically outlines an
MLPA reaction.
[0004] MLPA is particularly suitable for detecting nucleic acid
(pseudo)gene variants, (pseudo)gene-specific nucleotides and/or
copy number variation. MLPA has been employed in several studies,
e.g. for the diagnosis of Prader-Willi or Angelman syndromes, for
prenatal diagnosis of chromosomal aberrations in fetuses, and for
the detection of exon deletions and/or duplications in the Duchenne
muscular dystrophy gene. Overall, the conclusion was that MLPA
could replace the existing methods used for screening of
chromosomal abnormalities due to its relative simplicity,
reproducibility and speed.
[0005] In an MLPA assay, targeted nucleic acid which is
gene-specific or pseudogene-specific is preferably present at the
ligation site of the half probes. When a gene-specific or
pseudogene-specific nucleotide is present at (or within three
nucleotides from) a ligation site, this will ensure that only
perfectly matched half probes are ligated to each other. A mismatch
of a half probe at the ligation site prevents ligation and
amplification, whereas a perfect match of the half probe at the
ligation site allows ligation and amplification. As said before,
this allows MLPA to discriminate between sequences that only differ
in a single nucleotide. Mismatches at four to six nucleotides away
from the ligation site have been reported to have little effect on
the ligation step.
[0006] Hence, the half probes are preferably designed such that the
half probe whose 3' end hybridizes at a target sequence (called
herein a "left probe" or a "left half probe") is complementary to a
gene-specific sequence or pseudogene-specific sequence of the
target sequence. This gene-specific or pseudogene-specific sequence
of the target sequence comprises at least one but preferably more
nucleotides that make the probe specific for a given gene or
pseudogene. Preferably, at least one of the 3' end nucleotides of
said left half probe is complementary to at least one gene-specific
nucleotide and/or at least one pseudogene-specific nucleotide of
the target sequence, so that the (pseudo)gene-specific
nucleotide(s) or a single nucleotide polymorphism within a given
(pseudo)gene is present at (or within three nucleotides from) the
ligation site of said left half probe. In this case, said left half
probe and the probe whose 5' end hybridizes at a target sequence
(called herein a "right probe" or a "right half probe") are ligated
to each other only when the sequence of the left half probe
perfectly matches its target sequence.
[0007] As used herein the term "gene-specific nucleotide" or
"gene-specific sequence" means a nucleotide or sequence,
respectively, which is present in said gene but not present at the
corresponding location in at least one other related gene or
pseudogene. The term "pseudogene-specific nucleotide" or
"pseudogene-specific sequence" means a nucleotide or sequence,
respectively, which is present in said pseudogene but not present
at the corresponding location in at least one other related gene or
pseudogene. Hence, at least one other (pseudo)gene comprises
another nucleotide or sequence at that location. The presence of a
(pseudo)gene-specific nucleotide or (pseudo)gene-specific sequence
in a (pseudo)gene thus distinguishes said (pseudo)gene from at
least one other (pseudo)gene, even in case when the other
(pseudo)gene has a high overall homology with said
(pseudo)gene.
[0008] A pseudogene is defined herein as a nucleic acid sequence
which does not encode a wild type, functional, protein. The term
"pseudogene" encompasses nucleic acid sequences which do not encode
protein at all. Additionally, the term "pseudogene" encompasses
gene alleles which comprise a modification, for instance an
insertion or deletion so that they encode a protein or a part of a
protein with significantly impaired, or lost, function as compared
to a wild type protein of the same kind. Such allele for instance
encodes a truncated protein as a result of a frame shift caused by
an insertion and/or deletion of at least one nucleotide, or caused
by a premature stop codon.
[0009] Since ligases only ligate half probes which are adjacent to
each other, half probes need to be designed which are capable of
hybridizing immediately adjacent to each other on their target
sequence. This is not always convenient, because the hybridization
location of a left half probe on a target nucleic acid is often
determined by a (pseudo)gene-specific site of the target nucleic
acid (as explained above). In such case, the sequence of the
corresponding right half probe is determined as well, since the
right half probe should be capable of hybridizing to a region of
said target nucleic acid which is immediately adjacent to said
(pseudo)gene-specific nucleotide. However, such region may comprise
sequences which are very commonly present in the nucleic acid
sequences of a sample. As a result, a right half probe having a
sequence which is complementary to such common sequence will
hybridize at many different sites of the nucleic acids present in a
sample. In such case, it would be more attractive to design a right
half probe with a sequence which is more specific for a given site
of interest of a target nucleic acid. However, if the left half
probe and the right half probe do not hybridize to adjacent regions
of a target nucleic acid, the commonly used ligases will not be
capable of performing the ligation reaction. Patent application WO
01/61033 in the name of Schouten discloses a solution to this
problem by adding a short third probe to the reaction mixture,
which third probe will fill the gap between the left half probe and
the right half probe. Such third probe is designed to hybridize to
a region of a target nucleic acid which lies between the left and
the right half probes. After hybridization of such third probe, the
left half probe is connected to the right half probe via the third
probe and ligation has become possible. The third half probe does
not need to be perfectly complementary to the region of the target
nucleic acid which lies between the left and the right half probes,
as long as the third probe connects the left half probe and the
right half probe so that a ligase reaction can occur. Moreover,
since the third probe is small, it will hybridize more easily to
the target nucleic acid as compared to the left and right half
probes. Hence, mismatches between the third probe and the target
nucleic acid are allowed. This way, one and the same third probe is
suitable for connecting left and right half probes of different
probe sets.
[0010] Instead of using a third probe, WO 01/61033 also discloses
an embodiment wherein the 3' end of a left half probe is extended
after hybridization of the half probes to the target sequence, so
that the gap between the left half probe and the right half probe
is filled. The resulting extended left half probe is adjacent to
the right half probe and a ligase reaction has become possible.
[0011] In order to be capable of distinguishing between
amplificates of different probe sets, currently used MLPA probe
sets are designed such that the resulting amplificates have a
different length. Differences in ligated probe length are typically
realized by using a non-hybridizing stuffer sequence in one of the
half probes. The stuffer sequence of the half probes of each probe
set is unique in length, resulting in different lengths of
amplification products that can be separated by electrophoresis.
Typically, in order to be capable of discriminating between the
different amplification products, the difference in length between
different ligated probes is at least 5 nucleotides. Since a usual
MLPA assay involves the use of many different probe sets in order
to be capable of detecting a wide variety of (pseudo)gene variants,
this means that long probes have to be generated. This is
especially the case when complex loci carrying many
(pseudo)gene-specific nucleotides are investigated for proper
genotyping and/or additional single nucleotide polymorphisms are
investigated for detection of subtle genetic variation within a
specific genotype, as well as the presence of pseudogenes and
single nucleotides in these pseudogenes. Such investigation
requires the use of many different probe sets. This is inconvenient
if probes are chemically synthesized, because a drawback of
synthetic probes is the lower quality in comparison with cloned
probes, due to contamination with incompletely synthesized probes.
These incompletely synthesized probes lack or gain one nucleotide,
which results in stutter peaks and split peaks. A method to remove
these contaminants is to purify the synthesized probes, for
instance by polyacrylamid gel electrophoresis (PAGE). If short and
long probes are chemically synthesized, a higher proportion of
longer probes is more likely to be affected by the incomplete
oligonucleotides, causing a limitation of synthetic probe size. The
upper limit of synthetic probes is typically about 100 base
pairs.
[0012] On the other hand, the use of synthetic probes is preferred
because they are easy to obtain and cost-effective whereas
generating a probe by cloning in bacteriophage vectors is a
time-consuming process and more expensive.
[0013] Hence, although good results have been obtained with
currently used MLPA assays, it is desirable to provide alternatives
and improvements, especially if complex (pseudo)gene loci are
investigated which involves the use of many probe sets.
[0014] It is an object of the present invention to provide
alternative and improved MLPA methods and MLPA-like methods.
[0015] Accordingly, the present invention provides MLPA assays and
MLPA-like assays wherein at least one probe set is used which
comprises a first nucleic acid probe ("left probe" or "left probe
part"), a second nucleic acid probe ("right probe" or "right probe
part") and a third nucleic acid probe ("third probe" or "middle
probe" or "middle probe part"), wherein at least one third probe is
complementary to a target nucleic acid region comprising a
(pseudo)gene-specific nucleotide or (pseudo)gene-specific
sequence.
[0016] The present invention provides a different approach as
compared to the prior art. MLPA methods and MLPA-like methods are
now provided wherein at least one third probe, but preferably a
plurality of third probes, is used in order to detect at least one
(pseudo)gene-specific nucleotide of a target nucleic acid. Hence,
an additional probe is used in at least one of the probe sets,
which is specific for a (pseudo)gene-specific target nucleic acid.
As used herein, an MLPA-like method is defined as a method
comprising the steps of hybridisation of at least two probes to a
target nucleic acid and ligation of at least two probes.
Preferably, said MLPA-like method comprises amplification of
ligated probes as well.
[0017] MLPA methods and MLPA-like methods according to the present
invention have several advantages as compared to current methods.
For instance, if the left probe and the third probe of a probe set
are both complementary to target nucleic acid regions comprising
(pseudo)gene-specific nucleotides and/or additional single
nucleotide polymorphism(s), two different (pseudo)gene-specific
target nucleotides or two SNP's or a combination of one
(pseudo)gene specific target nucleotide and one SNP are screened
using one probe set. It has become possible to use one probe set in
order to screen for at least two (pseudo)gene variations which are
located within a region of about 150 nucleotides of a target
nucleic acid. Contrary, in a currently used MLPA assay two separate
probe sets are needed for screening for two variants in a target
nucleic acid. This is illustrated by the following example. If a
target (pseudo)gene contains a (pseudo)gene variant at location A
and at location B, an individual may comprise the following
alleles: a-b, a-B, A-b and A-B. In order to determine whether
allele a-B is present in a sample of said individual, a currently
used MLPA assay would need a probe set specific for the "a" and/or
"A" (pseudo)gene variant and a probe set specific for the "B"
and/or "b" (pseudo)gene variant. If both the probe set specific for
"a" and the probe set specific for "B" provide a positive result,
it is concluded that allele a-B is present in said individual. With
a MLPA method according to the present invention, however, only one
probe set is needed wherein the left probe is specific for the "a"
(pseudo)gene variant and the third probe is specific for the "B"
(pseudo)gene variant. If an amplification product is obtained, it
is immediately concluded that allele a-B is present in said
individual. If allele a-B is not present, said probe set according
to the invention will not yield an amplification product. Hence, it
has become possible to more specifically screen for a given
allele.
[0018] Moreover, a method of the invention provides an additional
advantage when two (pseudo)gene variations are located close to
each other. If the (pseudo)gene variants at location A and at
location B are close to each other, the use of two different probe
sets according to conventional MLPA techniques is inconvenient or
even not possible at all, because the two probe sets will hinder
each other in view of their close proximity. This will result in
less efficient hybridization of the two probe sets, resulting in a
lower signal as compared to a method according to the invention,
wherein two (pseudo)gene variants can be detected using only one
probe set. Hence, a method according to the invention is more
sensitive when (pseudo)gene variants are located close to each
other (in practice, this effect will be most profound when the
(pseudo)gene variants are located between 20-100 nucleotides from
each other). Having two probes to detect a variant at the same
position (such as in currently used MLPA assays) will result in a
change in signal intensity, depending on the presence of the
(pseudo)gene variant and the binding of the probe. The use of more
than two probes for one position is not advised. FIG. 1B
schematically outlines an MLPA reaction according to the invention
in which a probe set consisting of three probes is used for
detecting two SNPs. FIG. 1C shows a non-limiting example of two
specific probe sets according to the invention for detecting two
SNPs.
[0019] As another example, in case that an individual is
heterozygous for the above mentioned (pseudo)gene, the individual
for instance contains alleles a-B and A-b. A conventional MLPA
assay would use four probe sets (one specific for "a", one specific
for "A", one specific for "b" and one specific for "B"). Four
positive results would be obtained, because all four probe sets
would hybridize and result in an amplification product. However, in
such case it would still be unknown whether the individual
comprises the alleles a-b and A-B, or the alleles a-B and A-b. With
a method according to the present invention, however, it has become
possible to directly identify the alleles of said individual. For
instance, a first probe set of the invention is used comprising a
left probe specific for "a" and a third probe specific for "b",
together with a second probe set of the invention comprising a left
probe specific for "a" and a third probe specific for "B" and a
third probe set of the invention comprising a left probe specific
for "A" and a third probe specific for "b" and a fourth probe set
of the invention comprising a left probe specific for "A" and a
third probe specific for "B". Two of these probe sets according to
the present invention will yield an amplification product, namely
the second probe set of the invention comprising a left probe
specific for "a" and a third probe specific for "B" and the third
probe set of the invention comprising a left probe specific for "A"
and a third probe specific for "b". The first and fourth probe sets
according to the present invention will not yield (significant)
amplification product. This way, it is immediately apparent which
alleles are present in said individual. This, too, is an advantage
as compared to currently used methods, especially when complex loci
with many (pseudo)gene-specific nucleotides and additional single
nucleotide polymorphisms within a given (pseudo)gene are
investigated, because in such case many different combinations of
such (pseudo)gene variants need to be screened for.
[0020] Another advantage of a method according to the present
invention is the fact that more variations in length of the ligated
probes are obtained. Since at least one probe set of the invention,
but preferably a plurality of probe sets of the invention, comprise
a third probe it has become possible to design the probe sets such
that variations in length of the resulting ligated probes are
obtained. This obviates the need of stuffer sequences. As a result,
the individual probes of a probe set according to the invention can
be kept shorter, which is particularly advantageous when chemically
synthesized probes are used because chemical production of long
probes is cumbersome, as explained above. Hence, a method according
to the invention allows for the use of probe sets with relatively
short probes, while the resulting ligated probes are long enough to
allow for many size variations. Thus, the present invention allows
the use of synthetic probes, which are easy to obtain and
cost-effective, even when complex loci are investigated, and offers
greater flexibility to adapt the assay in case of cross-reactivity
or unclear results.
[0021] For instance, if 20 (pseudo)gene variants are investigated,
probes with a stuffer sequence with a length varying from 4 to 100
nucleotides would need to be used in a conventional MLPA assay in
order to be capable of distinguishing the resulting amplification
products by size. Since the probe sequences hybridizing to a target
sequence are typically about 30 nucleotides, and since the primer
binding sequences of the probes are typically about 15-25
nucleotides, this would mean that probe sets with probes with a
length varying from 45-125 nucleotides would need to be
synthesized. When the probes are chemically synthesized, it is
hardly possible to obtain reliable probe sets with these lengths.
With a method according to the invention, however, differences of
length between the various amplificates need not to be obtained by
use of stuffer sequences in the probe sets. Instead, at least one
third probe is used, preferably a plurality of third probes is
used. By varying combinations of three probes, optionally in
combination with probe sets consisting of two probes, the overall
length differences of the ligated probes vary considerably whereas
probe sets can be used with chemically synthesized probes with
convenient lengths. Of course, this does not mean that the use of
stuffer sequences is excluded. But the skilled person does no
longer have to rely on these stuffer sequences only for length
variations. If stuffer sequences are used in a method according to
the invention, it is preferred to keep these sequences as short as
possible.
[0022] Accordingly, the present invention provides a method for
screening for the presence of at least one target nucleic acid
sequence in a sample, comprising the steps of: [0023] a) adding to
said sample at least two different probe sets, each probe set
comprising: [0024] a first nucleic acid probe ("left probe"), said
first probe comprising a first nucleic acid sequence complementary
to a first region of said target nucleic acid sequence, and [0025]
a second nucleic acid probe ("right probe"), said second probe
comprising a second nucleic acid sequence complementary to a second
region of said target nucleic acid sequence, [0026] wherein at
least one of said probe sets comprises a third nucleic acid probe,
said third probe comprising a third nucleic acid sequence
complementary to a third region of said target nucleic acid
sequence, and [0027] wherein, if said third probe is present in
said probe set, said first and said third region of said target
nucleic acid are located essentially adjacent to each other and
said third and said second region of said target nucleic acid are
located essentially adjacent to each other, and [0028] wherein, if
said third probe is not present in said probe set, said first and
said second region of said target nucleic acid are located
essentially adjacent to each other, [0029] b) allowing
hybridization of said at least two different probe sets to
complementary nucleic acid of said sample, [0030] c) subjecting
nucleic acid of said sample to a ligation reaction, and [0031] d)
determining whether said at least one target nucleic acid sequence
is present in said sample, wherein at least one third nucleic acid
probe is complementary to a target nucleic acid region comprising a
(pseudo)gene variation.
[0032] The advantage of probe sets comprising at least three probes
according to the present invention is that at least two different
SNPs can be detected with one probe set. For instance, in a probe
set comprising three probes two sites for ligation are present. A
left probe and middle probe are ligated, and a middle probe and
right probe are ligated. At each ligation site a SNP can be
detected. Thus it is possible to design two probes of the same
probe set in such a way that they are used to detect two SNPs. In
that case, using MLPA and a probe set comprising three probes
according to the invention, a product will only be obtained when
both SNPs are present in a sample, because only then ligation can
occur at both ligation sites.
[0033] With conventional MLPA probesets consisting of two probes
only one SNP can be detected, because only one site for ligation is
present. Additional third probe parts in conventional MLPA, as
described in WO 01/61033, are occasionally used to bridge the two
half probes. Such an additional third probe part is not
SNP-specific. Therefore, the advantages of probe sets comprising at
least three probes according to the present invention are not
obtained when using such additional third probe part for bridging
purposes in conventional MLPA.
[0034] Therefore, in a preferred embodiment of the invention a
probe set comprises three nucleic acid probes wherein each of at
least two nucleic acid probes are specific for a different
(pseudo)gene variation. Preferably, a first (or a second) nucleic
acid probe of a probe set according to the invention is
complementary to a target nucleic acid region comprising a
gene-specific nucleotide and/or a pseudogene-specific nucleotide
and/or a gene-specific sequence and/or a pseudogene-specific
sequence and/or a polymorphism within a given gene or pseudogene,
and a third nucleic acid probe of the same probeset is
complementary to another target nucleic acid region comprising a
gene-specific nucleotide and/or a pseudogene-specific nucleotide
and/or a gene-specific sequence and/or a pseudogene-specific
sequence and/or a polymorphism within a given gene or pseudogene.
Said polymorphism preferably comprises an SNP.
[0035] Preferably, ligated probes are amplified. Accordingly, the
present invention provides a method for screening for the presence
of at least one target nucleic acid sequence in a sample,
comprising the steps of: [0036] a) adding to said sample at least
two different probe sets, each probe set comprising: [0037] a first
nucleic acid probe ("left probe"), said first probe comprising a
first nucleic acid sequence complementary to a first region of said
target nucleic acid sequence and, located 5' thereof, a
non-complementary nucleic acid sequence comprising a first primer
binding site, and [0038] a second nucleic acid probe ("right
probe"), said second probe comprising a second nucleic acid
sequence complementary to a second region of said target nucleic
acid sequence and, located 3' thereof, a non-complementary nucleic
acid sequence comprising a second primer binding site, [0039]
wherein at least one of said probe sets comprises a third nucleic
acid probe, said third probe comprising a third nucleic acid
sequence complementary to a third region of said target nucleic
acid sequence, and [0040] wherein, if said third probe is present
in said probe set, said first and said third region of said target
nucleic acid are located essentially adjacent to each other and
said third and said second region of said target nucleic acid are
located essentially adjacent to each other, and [0041] wherein, if
said third probe is not present in said probe set, said first and
said second region of said target nucleic acid are located
essentially adjacent to each other, [0042] b) allowing
hybridization of said at least two different probe sets to
complementary nucleic acid of said sample, [0043] c) subjecting
nucleic acid of said sample to a ligation reaction, [0044] d)
subjecting nucleic acid of said sample to a nucleic acid
amplification reaction, using at least one primer capable of
specifically binding said first primer binding site and at least
one primer capable of specifically binding said second primer
binding site, and [0045] e) determining whether amplified nucleic
acid is present, thereby determining whether said at least one
target nucleic acid sequence is present in said sample, [0046]
wherein at least one third nucleic acid probe is complementary to a
target nucleic acid region comprising a (pseudo)gene variation.
[0047] As used herein, the term "(pseudo)gene variation"
encompasses a (pseudo)gene-specific nucleotide and/or a
(pseudo)gene-specific sequence. In one embodiment, said
(pseudo)gene variation comprises an additional polymorphism within
a given (pseudo)gene. Said additional polymorphism preferably
comprises an SNP.
[0048] Hence, the present invention uses probe sets, wherein at
least one probe set, but preferably a plurality of probe sets,
comprises three probes. The probes comprise sequences which are
complementary to a region of a target nucleic acid of interest. As
used herein, the term "complementary" means that said probe
sequence comprises at least 70%, preferably at least 80%, more
preferably at least 85%, more preferably at least 90%, most
preferably at least 95% sequence identity to said region or to the
complement of said region. The term "% sequence identity" is
defined herein as the percentage of residues in a nucleotide
sequence that is identical with the residues in a reference
sequence after aligning the two sequences and introducing gaps, if
necessary, to achieve the maximum percent identity. Methods and
computer programs for the alignment are well known in the art. One
computer program which may be used or adapted for purposes of
determining whether a candidate sequence falls within this
definition is Autoassembler 2.0 (ABI Prism, Perkin Elmer).
[0049] The first and second probes of each probe set also comprise
a primer binding site, so that the resulting ligated probes can be
amplified. Preferably, the primer binding sites of the first
nucleic acid probes of each probe set is designed such that the
same primer can bind. This allows the use of the same primer for
binding the primer binding sites of the first probes in step d).
Likewise, it is preferred that the primer binding sites of the
second nucleic acid probes of each probe set is designed such that
the same primer can bind. Most preferably, the probe sets are
designed such that a first primer is capable of specifically
binding the primer binding sites of the first nucleic acid probes
of each probe set and a second primer is capable of specifically
binding the primer binding sites of the second nucleic acid probes
of each probe set. This embodiment allows the use of only one
primer pair in step d). This is, however, not necessary: it is also
possible to use different primers for different probe sets. The
number of different primers is, however, kept as low as
possible.
[0050] One preferred embodiment therefore provides a method
according to the invention, wherein the first primer binding sites
of the first nucleic acid probes of each probe set is capable of
specifically binding the same primer and/or wherein the second
primer binding sites of the second nucleic acid probes of each
probe set is capable of specifically binding the same primer.
Preferably, the first nucleic acid probes and/or the second nucleic
acid probes of each probe set comprise essentially identical primer
binding sequences. Further provided is therefore a method according
to the invention, wherein the non-complementary nucleic acid
sequences of said first nucleic acid probes comprise essentially
identical first primer binding sites and/or wherein the
non-complementary nucleic acid sequences of said second nucleic
acid probes comprise essentially identical second primer binding
sites. Using essentially identical primer binding sequences ensures
that the same primer can bind different probes. The term
"essentially identical primer binding sequences" is defined herein
as primer binding sequences which comprise at least 80%, preferably
at least 85%, more preferably at least 90%, most preferably at
least 95% sequence identity to each other.
[0051] As already described, a method according to the invention is
particularly suitable for investigating a nucleic acid sequence
having various (pseudo)gene specific nucleotides and/or
(pseudo)gene variants, such as complex loci. It is therefore
preferred to use a plurality of third probes, so that many
(pseudo)gene variant combinations are investigated. A method
according to the invention is therefore preferably provided wherein
at least two, preferably at least five, more preferably at least
ten different third nucleic acid probes are used. As illustrated in
the Examples, a plurality of probe sets comprising different third
probes according to the invention allows for screening of complex
gene loci such as the KIR locus. Not all third probes need to be
specific for a genetic variation of a target nucleic acid. It is
also possible to use a combination of variant-specific third probes
and third probes which are not specific for a (pseudo)gene
variation. Likewise, not all first probes need to be specific for a
variant of a target nucleic acid. It is also possible to use a
combination of variant-specific first probes and first probes which
are not specific for a (pseudo)gene variation. Any of these
combinations is for instance used to vary the length of the
resulting ligated probes to a larger extent. In one preferred
embodiment of the invention, therefore, at least 50%, preferably at
least 70%, more preferably at least 80%, most preferably at least
90% of the third nucleic acid probes is complementary to a target
nucleic acid region comprising a (pseudo)gene variation. In one
embodiment, all third probes are complementary to a target nucleic
acid region comprising a (pseudo)gene variant. Preferably, the
second probes ("right probes") are not designed to contain
(pseudo)gene variant-specific sequences, although the use of
variant-specific right probes in a method according to the
invention is not excluded.
[0052] Preferably, at least 50%, preferably at least 70%, more
preferably at least 80%, most preferably at least 90% of the third
nucleic acid probes that are complementary to a target nucleic acid
region comprising a (pseudo)gene variation are combined with a
first nucleic acid probe or a second nucleic acid probe that is
complementary to another target nucleic acid region comprising a
(pseudo)gene variation in order to be capable of screening for many
variants with one MLPA assay or MLPA-like assay. In one embodiment,
all third probes that are combined with a first nucleic acid probe
or a second nucleic acid probe that is complementary to a target
nucleic acid region comprising a (pseudo)gene variation are
complementary to a target nucleic acid region comprising a
(pseudo)gene variant. Of course, these probes are preferably
specific for different variants.
[0053] In one preferred embodiment, a (pseudo)gene variant-specific
sequence of a third probe is at least located within the last three
nucleotides or the first three nucleotides of the third probe. This
means that the last three nucleotides and/or the first three
nucleotides comprise at least one nucleotide which is specific for
a (pseudo)gene variation of a target nucleic acid. In this
embodiment, said (pseudo)gene variation is present at a ligation
site of the third probe, so that ligation is only possible when the
sequence of the third probe is exactly complementary to said
(pseudo)gene variation. This enhances the specificity of the MLPA
method, as explained before. Preferably, the last three nucleotides
and/or the first three nucleotides of said third probe comprise one
nucleotide which is specific for a (pseudo)gene variant of a target
nucleotide.
[0054] The probe sets according to the present invention preferably
have a length between 90 and 300 nucleotides. Cloned probes can be
as long as 500 nucleotides. Preferably, however, chemically
synthesized probes are used because they are rapidly synthesized,
easy to obtain and cost-effective. In order to be capable of
synthetically producing the probes according to the present
invention, a method according to the invention is preferably
provided wherein third nucleic acid probes with a length of between
20 and 100 nucleotides are used. Most preferably, third nucleic
acid probes with a length of between 19 and 110 nucleotides are
used. Since at least one probe set of the invention, but preferably
a plurality of probe sets according to the invention, is used which
comprise three nucleic acid probes, sufficient variations in length
and specificity of the resulting ligated probes is ensured so that
many (pseudo)gene variations can be investigated
simultaneously.
[0055] These length variations of the resulting ligated probes
obviate the need of stuffer sequences, as explained before. It is
therefore possible to design the probe sets such that the parts of
the first and/or second probe which are not complementary to a
target nucleic acid have about the same length. According to this
embodiment, the length of the non-complementary sequences of all
first probes is about the same in each probe set, and/or the length
of the non-complementary sequences of all second probes is about
the same in each probe set. These lengths are about the same when
they do not differ from each other by more than 10 nucleotides.
Preferably, they do not differ from each other by more than 6
nucleotides, most preferably they do not differ from each other by
more than 4 nucleotides. This, too, facilitates synthetic
production of the probes. Further provided is therefore a method
according to the invention, wherein the difference in length of
said non-complementary nucleic acid sequences of said first nucleic
acid probes of said at least two different probe sets and/or the
difference in length of said non-complementary nucleic acid
sequences of said second nucleic acid probes of said at least two
different probe sets is less than 6, preferably less than 4 nucleic
acids.
[0056] Besides the analysis of (pseudo)gene-specific nucleotides
and additional single nucleotide polymorphisms, an MLPA technique
or MLPA-like technique is particularly suitable for relative
(pseudo)gene copy number determination. If multiple copies of a
(pseudo)gene of interest (or any other target nucleic acid of
interest) are present in sample nucleic acid molecules, each copy
will, in principle, be bound by the specific probes which is
detectable. When the probes are amplified, more amplification
product will be present when multiple copies were present in the
original sample nucleic acid as compared to a situation wherein
only one copy is present. Analysis of the amount of amplification
product thus provides information about the copy number of a target
nucleic acid of interest. This is often done by graphically
representing amplified products by separate peaks. Each peak is the
product of an amplified MLPA ligated probe and a relative
difference in peak intensity (height or surface) between a control
sample and a sample of interest indicates copy number variation.
When a complex locus is investigated, multiple copies of a
(pseudo)gene of interest can be present in highly polymorphic
regions. In such case, when (pseudo)gene copy number is to be
determined, many different combinations of (pseudo)gene variants
need to be taken into account. This involves the use of a wide
variety of different probe sets, to ensure that each combination of
(pseudo)gene variants can be detected. In one embodiment according
to the present invention, however, when the relative copy number of
a nucleic acid of interest is to be estimated, an improved approach
is provided. According to this embodiment, at least one probe is
used with degenerate bases at one or more positions. This means
that a mixture of probes is used wherein different nucleotides can
be present at one or more positions. Hence a mixture of probes is
used, which probes have the same sequence, except for the fact that
some probes have a certain nucleotide at a given position X and
some probes have another nucleotide at said position X. Such
degenerate bases are commonly represented by the IUB nucleotide
codes as depicted in FIG. 2. The use of probes with degenerate
bases allows for an efficient estimation of copy number of a
nucleic acid of interest, even in highly polymorphic regions.
Further provided is therefore a method for determining the copy
number of a nucleic acid of interest, wherein at least one probe
set is used which comprises a probe with (a) degenerate base(s) at
one or more positions. Preferably, at most 20 probe positions have
such multiple alternatives, in order to retain specificity of the
probes for a given target region of interest. A use of at least one
probe set for determining the copy number of a nucleic acid of
interest, wherein at least one probe set comprises a probe with (a)
degenerate base(s) at one or more positions, is also provided
herewith. In one preferred embodiment, at least one probe set
comprising a probe with (a) degenerate base(s) is used in a MLPA
method or MLPA-like method according to the present invention.
Further provided is therefore a method according to the invention,
wherein at least one probe set is used which comprises a probe with
(a) degenerate base(s) at one or more positions.
[0057] Alternatively, or additionally, a probe set is used which
comprises an alternative base which alternative base is capable of
binding at least two bases selected from the group consisting of A,
T, G, C and U. Preferably, said alternative base is capable of
binding at least three, most preferably at least four, bases
selected from the group consisting of A, T, G, C and U. Such
alternative base is suitable as an alternative for degenerate
bases. It is, of course, also possible to combine such alternative
base with degenerate bases. In a particularly preferred embodiment
said alternative base is deoxyinosine triphosphate (dITP) or a
functional equivalent thereof, which is capable of binding A and T
and G and C and U. Further provided is therefore a method for
determining the copy number of a nucleic acid of interest, wherein
at least one probe set is used which comprises an alternative base
which is capable of binding at least two, preferably at least
three, more preferably at least four bases selected from the group
consisting of A, T, G, C and U. As said before, said alternative
base preferably comprises deoxyinosine triphosphate (dITP) or a
functional equivalent thereof. A use of at least one probe set for
determining the copy number of a nucleic acid of interest, wherein
at least one probe set comprises an alternative base which is
capable of binding at least two, preferably at least three, more
preferably at least four bases selected from the group consisting
of A, T, G, C and U, is also provided herewith. In one preferred
embodiment, at least one probe set comprising such alternative
base(s) is used in a MLPA method or MLPA-like method according to
the present invention. Further provided is therefore a method
according to the invention, wherein at least one probe set is used
which comprises an alternative base which is capable of binding at
least two, preferably at least three, more preferably at least four
bases selected from the group consisting of A, T, G, C and U. As
said before, said alternative base preferably comprises
deoxyinosine triphosphate (dITP) or a functional equivalent
thereof.
[0058] The present invention provides alternative and improved
methods for screening for the presence of at least one target
nucleic acid sequence in a sample, wherein at least one third probe
is used which is complementary to a target nucleic acid region
comprising a (pseudo)gene variation. A use of a probe set
comprising at least three nucleic acid probes, wherein at least one
third probe is complementary to a target nucleic acid region
comprising a gene variant and/or a pseudogene variant, for
screening for the presence of at least one target nucleic acid
sequence in a sample is therefore also provided. Preferably, a
plurality of probe sets according to the present invention is used.
Further provided is therefore a use of a plurality of probe sets
for screening for the presence of at least one target nucleic acid
sequence in a sample, wherein each of said probe sets comprises:
[0059] a first nucleic acid probe, said first probe comprising
[0060] a first nucleic acid sequence complementary to a first
region of said target nucleic acid sequence and, located 5'
thereof, a non-complementary nucleic acid sequence comprising a
first primer binding site, and [0061] a second nucleic acid probe,
said second probe comprising [0062] a second nucleic acid sequence
complementary to a second region of said target nucleic acid
sequence and, located 3' thereof, a non-complementary nucleic acid
sequence comprising a second primer binding site, [0063] wherein at
least one of said probe sets comprises a third nucleic acid probe,
said third probe comprising a third nucleic acid sequence
complementary to a third region of said target nucleic acid
sequence, and [0064] wherein, if said third probe is present in
said probe set, said first and said third region of said target
nucleic acid are located essentially adjacent to each other and
said third and said second region of said target nucleic acid are
located essentially adjacent to each other, and [0065] wherein, if
said third probe is not present in said probe set, said first and
said second region of said target nucleic acid are located
essentially adjacent to each other, and [0066] wherein at least one
third nucleic acid probe is complementary to a target nucleic acid
region comprising a gene-specific nucleotide and/or a
pseudogene-specific nucleotide and/or a gene-specific sequence
and/or a pseudogene-specific sequence and/or an additional
polymorphism within a given gene or pseudogene, said polymorphism
preferably comprising an SNP.
[0067] A method according to the present invention is particularly
suitable for analysis of (pseudo)gene variation and (pseudo)gene
copy number determination in complex loci such as the gene encoding
complement factors (e.g. Factor H and FH-like genes, C4A and C4B
within the HLA-class III region), chemokines and their receptor
alleles (e.g. CCL3L1, CCL4L1, CCR5 or CCR5delta32), HLA-class I and
II, SIRPs and LILRs.
[0068] In one preferred embodiment, a method according to the
invention is used in order to investigate the killer cell
immunoglobulin-like receptor (KIR) locus. KIRs are expressed by
natural killer (NK) cells and a subset of T cells. NK cells are
cells of the lymphoid lineage, but display no antigen-specific
receptors. Their main function is to monitor host cells for the
presence of MHC class I molecules and this is important for e.g.
distinguishing healthy cells from virus-infected or tumors cells.
Interaction between NK cells and MHC class I molecules is mediated
by KIRs. The KIR locus in humans is polygenic and highly
polymorphic, so that accurate and efficient characterization of an
individual's KIR (pseudo)gene profile is cumbersome. In the
determination of the KIR (pseudo)gene profile and their role in
many diseases an efficient and reliable method for KIR genotyping
is, however, important. Until now, KIR genotyping is based upon the
polymerase chain reaction sequence-specific primer (PCR-SSP) (Sun
et al, 2004), multiplex PCR (Vilches et al, 2007) and PCR-sequence
specific oligonucleotide probes (PCR-SSOP) (Crum et al, 2000). For
the PCR-SSP high-quality genomic DNA is required and multiple
reactions are needed to generate a complete KIR profile of an
individual. Multiple copies of KIR2DL4 and KIR3DL1/S1 in
individuals have been reported with PCR-SSOP (Williams et al,
2003). Detection of the multiple gene copies was possible because
the gene copies of these genes consisted of different alleles.
However, multiple gene copies of highly homologous or identical
sequences are not distinguishable with this molecular detection
system or cloning methods when individuals are homozygous for a
gene (Williams et al, 2003).
[0069] As shown in the Examples, a method according to the present
invention is particularly suitable for investigating the KIR locus
of individuals. Even though this locus is highly polymorphic,
(pseudo)gene variants and copy number variations are efficiently
detected with methods according to the present invention. One
preferred embodiment therefore provides a method or use according
to the invention, wherein said target nucleic acid sequence is
present in a KIR locus. Preferably, copy number variation of at
least one KIR gene and/or at least one KIR pseudogene is
determined. FIGS. 3A and B provides KIR-specific probes which
provide particularly good results. These probes are therefore
preferred when a KIR locus is investigated. FIGS. 3C and D provides
an extended list of KIR-specific probes which provide even better
results than the probes listed in FIGS. 3A and B. Therefore, these
probes are even more preferred when a KIR locus is investigated.
Further provided is thus a method and/or a use according to the
invention, wherein at least one probe depicted in FIG. 3A, 3B, 3C
or 3D, preferably in FIG. 3C or 3D, is used. Preferably, at least
two probes depicted in FIG. 3 are used. In another preferred
embodiment at least four probes, more preferably at least six
probes depicted in FIG. 3A, 3B, 3C or 3D are used.
[0070] In a particularly preferred embodiment, a probe set of FIG.
3 is used. Said probe set preferably comprises three probes. A
probe set of FIG. 3 is formed by two or three individual probes
depicted in FIG. 3 which have the same number, followed by the
letter A, B, C, D, E, G, K, L, M or N. For instance, probe set 408
is formed by probes 408A, 408B and 408C. Optionally, four different
probes with the same number are given for a probe set of FIG. 3. In
that case, a left, a middle and a right probe is selected from said
four probes. Further provided is therefore a method and/or a use
according to the invention, wherein at least one probe set depicted
in FIG. 3A selected from the group consisting of probe set 408,
probe set 507, probe set 419, probe set 528, probe set 413, probe
set 416, probe set 415 and probe set 418 is used. In a particularly
preferred embodiment at least one probe set depicted in FIG. 3A
selected from the group consisting of probe set 408, probe set 507,
probe set 528, probe set 413, probe set 416 and probe set 415 is
used. These probe sets contain a third probe which is specific for
a (pseudo)gene variant of the KIR locus. Also provided is a method
and/or a use according to the invention, wherein at least one probe
set depicted in FIG. 3B selected from the group consisting of probe
set 409, probe set 506, probe set 507, probe set 538, probe set 417
and probe set 517 is used. In a particularly preferred embodiment
at least one probe set depicted in FIG. 3B selected from the group
consisting of probe set 409, probe set 506, probe set 507, probe
set 538, probe set 417 and probe set 517 is used. These probe sets
also contain a third probe which is specific for a (pseudo)gene
variant of the KIR locus. Also provided is a method and/or a use
according to the invention, wherein at least one probe set depicted
in FIG. 3C selected from the group consisting of probe set 415,
probe set 703, probe set 413, probe set 419, probe set 702, probe
set 711, probe set 408, probe set 507, probe set 710, probe set
528, probe set 418 and probe set 416 is used. In a particularly
preferred embodiment at least one probe set depicted in FIG. 3C
selected from the group consisting of probe set 415, probe set 703,
probe set 413, probe set 419, probe set 702, probe set 711, probe
set 408, probe set 507, probe set 710, probe set 528, probe set 418
and probe set 416 is used. These probe sets also contain a third
probe which is specific for a (pseudo)gene variant of the KIR
locus. Also provided is a method and/or a use according to the
invention, wherein at least one probe set depicted in FIG. 3D
selected from the group consisting of probe set 506, probe set 417,
probe set 517, probe set 409, probe set 507, probe set 710, probe
set 709, probeset 708, probe set 704 and probe set 538 is used. In
a particularly preferred embodiment at least one probe set depicted
in FIG. 3D selected from the group consisting of probe set 506,
probe set 417, probe set 517, probe set 409, probe set 507, probe
set 710, probe set 709, probeset 708, probe set 704 and probe set
538 is used. These probe sets also contain a third probe which is
specific for a (pseudo)gene variant of the KIR locus.
[0071] It is preferred to use at least two probe sets selected from
FIG. 3, so that various KIR (pseudo)gene variants are screened for
with good results. More preferably, at least three probe sets
selected from FIG. 3 are used. Even more preferably, at least four,
more preferably at least five, most preferably at least six probe
sets selected from FIG. 3 are used. Said at least two, three, four,
five or six probe sets are preferably selected from the group
consisting of probe set 408, probe set 507, probe set 528, probe
set 413, probe set 416, probe set 415, probe set 418, probe set
419, probe set 409, probe set 506, probe set 538, probe set 417,
probe set 517, probe set 703, probe set 702, probe set 711, probe
set 710, probe set 709 and probe set 704 since these probe sets
contain a third probe which is specific for a (pseudo)gene variant
of the KIR locus. In one embodiment, all probe sets depicted in
FIG. 3A, and/or 3B, and/or 3C, and/or 3D are used. In a preferred
embodiment all probe sets depicted in FIG. 3C and/or FIG. 3D are
used.
[0072] It is of course also possible to modify a sequence of at
least one probe depicted in FIG. 3 to some extent. This is for
instance done for optimalization purposes. Further provided is
therefore a method and/or a use according to the invention, wherein
at least one probe is used which has at least 70%, preferably at
least 80%, more preferably at least 85%, more preferably at least
90%, most preferably at least 95% sequence identity to a probe
depicted in FIG. 3. Preferably, at least two, more preferably at
least four, most preferably at least six probes are used which have
at least 70%, preferably at least 80%, more preferably at least
85%, more preferably at least 90%, most preferably at least 95%
sequence identity to a probe depicted in FIG. 3. In one embodiment,
a method or use according to the invention is provided wherein at
least 20 probes are used, said at least 20 probes having at least
70%, preferably at least 80%, more preferably at least 85%, more
preferably at least 90%, most preferably at least 95% sequence
identity to the probes depicted in FIG. 3. A minimum of two
specific probes per (pseudo)gene is preferred to determine copy
number variation (CNV).
[0073] Preferably, probe sets are used which are based on the probe
sets depicted in FIG. 3A, 3B, 3C or 3D, preferably based on the
probe sets depicted in FIG. 3C and/or 3D. Said probe set preferably
comprises three probes. One or more of the probes of such probe set
may be modified to some extent, as described above. Further
provided is therefore a method and/or a use according to the
invention, wherein at least one probe set is used which has at
least 70%, preferably at least 80%, more preferably at least 85%,
more preferably at least 90%, most preferably at least 95% sequence
identity to a probe set as depicted in FIG. 3. This means that the
probes of said probe set have at least 70% sequence identity to the
corresponding probes of at least one probe set of FIG. 3.
Preferably, a probe set is used which has at least 70%, preferably
at least 80%, more preferably at least 85%, more preferably at
least 90%, most preferably at least 95% sequence identity to a
probe set depicted in FIG. 3 selected from the group consisting of
probe set 408, probe set 507, probe set 419, probe set 528, probe
set 413, probe set 416, probe set 415, probe set 418, probe set
419, probe set 409, probe set 506, probe set 538, probe set 417,
probe set 517, probe set 703, probe set 702, probe set 711, probe
set 710, probe set 709 and probe set 704 since these probe sets
contain a third probe specific for a KIR nucleic acid sequence.
Preferably at least two, more preferably at least three, more
preferably at least four, more preferably at least five, most
preferably at least six of such probe sets are used, so that
various KIR (pseudo)gene variants are screened for with good
results.
[0074] Novel probes and probe sets which are particularly suitable
for (pseudo)gene variant analysis and (pseudo)gene copy number
determination of the KIR locus are also provided. These probes and
probe sets are listed in FIG. 3A, B, C and D, as described above.
Further provided are therefore probes and probe sets as depicted in
FIG. 3A, 3B, 3C or 3D, as well as probes and probe sets which have
at least 70%, preferably at least 80%, more preferably at least
85%, more preferably at least 90%, most preferably at least 95%
sequence identity to a probe or probe set depicted in FIG. 3A, 3B,
3C or 3D. A mixture of nucleic acids, wherein said nucleic acids
comprise at least two probe sets according to the invention is also
provided. Preferably, said mixture comprises at least four, more
preferably at least six probe sets according to the invention. As
said before, such probe sets have at least 70% sequence identity to
a probe or probe set depicted in FIG. 3A, 3B, 3C or 3D. One
embodiment provides a mixture of nucleic acids comprising at least
two, preferably at least four, more preferably at least six probe
sets as depicted in FIG. 3A, 3B, 3C or 3D.
[0075] Further provided is a kit for detecting the presence of at
least one target nucleic acid sequence in a sample, comprising a
probe set or a mixture of nucleic acids according to the invention.
Said at least one target nucleic acid sequence preferably comprises
a nucleic acid sequence present in a KIR locus. A kit according to
the invention preferably further comprises a PCR primer set
comprising at least 70%, preferably at least 80%, more preferably
at least 85%, more preferably at least 90%, most preferably at
least 95% sequence identity to nucleic acid sequences
5'-GGGTTCCCTAAGGGTTGGA and TCTAGATTGGATCTTGCTGGCAC-3', or the
complements thereof. These primers are particularly suitable for
amplifying probe sets depicted in FIG. 3.
[0076] KIR polymorphisms have been associated with disease.
Association between KIR polymorphisms and subtypes of leukemia were
investigated by Zhang et al. (Zhang et al. 2009). The presence of
KIR2DS4 was demonstrated to be predisposing to chronic myelogenous
leukemia (CML) and the absence of KIR2DS3 was predisposing to acute
lymphoblastic leukemia (ALL). KIR2DS4 is present in haplotype A,
whereas KIR2DS3 is present in haplotype B. Presence of KIR2DS4 and
absence of KIR2DS3 are predisposing to leukemia subtypes. Thus,
characteristics of haplotype A are predisposing to leukemia
subtypes. The present invention provides probes that are
particularly well suitable for detecting KIR genes, including
KIR2DS4 and KIR2DS3. Thus, with probes according to the present
invention selected from FIG. 3A, 3B, 3C and/or 3D the presence
and/or absence of KIR2DS4 and KIR2DS3 in a sample is particularly
well determined. Preferably probesets 540A/540C, and/or 513B/513D
and/or 504A/504B, and/or 708K/708L/708M/708N as depicted in FIG. 3C
and/or 3D are used to detect KIR2DS3 and/or KIR2DS4 polymorphisms.
With probes selected from FIG. 3 predisposition to leukemia
subtypes is thus particularly well determined.
[0077] Therefore, in one embodiment the invention provides a method
for determining predisposition to leukemia of an individual
comprising determining the presence or absence of KIR2DS4 and/or
KIR2DS3 in a nucleic acid sample of said individual with at least
one probeset listed in FIG. 3A, 3B, 3C and/or 3D, wherein the
presence of KIR2DS4 is indicative for a predisposition for chronic
myelogenous leukemia and the absence of KIR2DS3 is indicative for a
predisposition for acute lymphoblastic leukemia. In a preferred
embodiment probe set 540A/540C, and/or 513B/513D and/or probe set
504A/504B, and/or 708K/708L/708M/708N as depicted in FIG. 3C and/or
3D are used for determining the presence or absence of KIR
polymorphisms. As used herein, the term "nucleic acid sample" means
a sample comprising nucleic acid. Said sample may of course further
comprise other components, such as for instance proteins.
Preferably, nucleic acid is at least partly isolated from said
sample before being subjected to a method according to the present
invention.
[0078] Association between KIR polymorphisms and inflammatory bowel
disease (IBD) and/or Crohn's disease have been established as well
(Hollenbach et al 2009). The KIR2DL2/KIR2DL3 heterozygous genotype
predisposes or protects from Crohn's disease depending on the
presence of their HLA-C ligands. KIR2DL2/KIR2DL3 heterozygosity in
combination with C1 predisposes to Crohn's disease whereas
KIR2DL2/KIR2DL3 heterozygosity in combination with C2 protects from
IBD and/or Crohn's disease. KIR2DL2/KIR2DL3 heterozygosity in
combination with C1/C2 heterozygosity has an intermediate effect on
predisposition (Hollenbach et al 2009). Non-limiting examples for
determining the presence or absence of C1 and/or C2 are detecting
nucleic acid sequence(s) encoding C1 and/or C2 protein using for
instance a nucleic acid amplification reaction or detecting C1
and/or C2 protein using for instance Western blot analysis.
[0079] The present invention provides probes that are particularly
suitable for detecting KIR genes, including KIR2DL2 and KIR2DL3.
Thus, with probes according to the present invention selected from
FIG. 3A, 3B, 3C and/or 3D KIR2DL2/KIR2DL3 heterozygosity in a
sample is particularly well determined. Preferably probeset
415B/415C/415D and/or 417A/417B/417C and/or probeset 420A/420B,
and/or 706A/706B as depicted in FIG. 3C and/or 3D are used to
detect KIR2DL3 and/or KIR2DL2 polymorphisms. With probes selected
from FIG. 3 predisposition to Crohn's disease is thus particularly
well determined.
[0080] Therefore, in one embodiment the invention provides a method
for determining predisposition to IBD and/or Crohn's disease of an
individual comprising determining the presence or absence of
KIR2DL2 and/or KIR2DL3 in a nucleic acid sample of said individual
with at least one probeset listed in FIG. 3A, 3B, 3C and/or 3D, and
determining the presence of absence of HLA C1 and/or C2 ligand in a
sample of said individual, wherein KIR2DL2, KIR2DL3 heterozygosity
in combination with C1 homozygosity is indicative for a
predisposition for Crohn's disease, and KIR2DL2, KIR2DL3
heterozygosity in combination with C2 homozygosity is indicative
for protection for Crohn's disease. In a preferred embodiment probe
set 415B/415C/415D and/or 417A/417B/417C and/or probe set 420A/420B
and/or 706A/706B as depicted in FIG. 3C and/or 3D are used for
determining the presence or absence of KIR polymorphisms.
[0081] Copy number variation of KIR2DL3, KIR3DL1 and KIR3DS1 is
correlated to the course of disease in chronic infection, such as
retroviral infection, herpes virus infection, and hepatitis virus
infection, more in particular HIV, CMV, EBV, HSV, HBV and HCV
(Martin et al 2007 and Khakoo et al 2004). A higher copy number of
KIR3DL1 and/or KIR3DS1 in an individual is indicative for an
improved course of the disease and/or response to treatment of
chronic infection as compared with a low copy number of KIR3DL1
and/or KIR3DS1 in an individual and a low copy number of KIR2DL3 in
an individual is indicative for an improved course of the disease
and/or response to treatment of chronic infection as compared with
a high copy number of KIR2DL3 in an individual. Thus, a higher copy
number of KIR3DL1 and/or KIR3DS1 in an individual is indicative for
an increased survival in chronic infection and a lower copy number
of KIR2DL3 in an individual is indicative for increased survival in
chronic infection.
[0082] The present invention provides probes that are particularly
well suitable for determining copy number variation of KIR genes,
including KIR3DL1 and KIR3DS1. Thus, with probes according to the
present invention selected from FIG. 3A, 3B, 3C and/or 3D the copy
number of KIR3DL1 and KIR3DS1 and KIR2DL3 in a sample is
particularly well determined. Preferably probe sets 409A/409B/409C,
and/or 711A/711B/711C/711D and/or 418A/418B/418D, and/or
709C/709D/709E/709G and/or probe set 415B/415C/415D and/or
417A/417B/417C as depicted in FIG. 3C and/or 3D are used to
estimate the copy number of KIR3DL1 and/or KIR3DS1 and/or KIR2DL3.
With probes selected from FIG. 3 susceptibility of an individual to
course of disease and/or response to treatment in chronic infection
is thus particularly well determined.
[0083] Therefore the invention provides method for determining
susceptibility of an individual to course of disease and/or
response to treatment in chronic infection, preferably retroviral
infection, herpes virus infection, and hepatitis virus infection,
comprising determining the copy number of KIR2DL3, KIR3DL1 and/or
KIR3DS1 in a nucleic acid sample of said individual with at least
one probeset listed in FIG. 3A or 3B or 3C or 3D, wherein a high
KIR3DL1 and/or KIR3DS1 copy number in an individual is indicative
for an improved course of disease and/or response to treatment of
chronic infection as compared with a low copy number of KIR3DL1
and/or KIR3DS1 in an individual and a low KIR2DL3 copy number in an
individual is indicative for an improved course of disease and/or
response to treatment of chronic infection as compared with a high
copy number of KIR2DL3 in an individual. Preferably said chronic
infection comprises HIV, CMV, EBV, HSV, HBV and HCV. In a preferred
embodiment probeset 409A/409B/709D/409C, and/or 711A/711B/711C/711D
and/or 418A/418B/418D, and/or 709C/709E/709G and/or probe set
415B/415C/415D and/or 417A/417B/417C as depicted in FIG. 3C and/or
3D are used for determining the copy number of KIR genes.
[0084] The presence of KIR2DS4 in a donor is correlated to
transplantation-related outcome measures, such as mortality,
graft-versus-host, graft-versus-tumor and grafted organ survival in
recipients after transplantation. The presence of KIR2DS4 in a
donor is indicative for reduced mortality, reduced
graft-versus-host, increased graft-versus-tumor and increased
grafted organ survival in recipients after transplantation as
compared to the absence of KIR2DS4 in a donor. The present
invention provides probes that are particularly well suitable for
determining copy number variation of KIR genes, including KIR3DL1
and KIR3DS1. Thus, with probes according to the present invention
selected from FIG. 3A, 3B, 3C and/or 3D the copy number of KIR2DS4
in a sample is particularly well determined. Preferably probe sets
504A/504B, and/or 708K/708L/708M/708N as depicted in FIG. 3C and/or
3D are used to the presence or absence of KIR2DS4. With probes
selected from FIG. 3 predisposition to transplantation-related
outcome measures is thus particularly well determined
[0085] Therefore the invention provides a method for determining
predisposition to transplantation-related outcome measures, such as
mortality, graft-versus-host, graft-versus-tumor and grafted organ
survival of a recipient after transplantation, comprising
determining the presence or absence of KIR2DS4 in a nucleic acid
sample of a donor for said recipient with at least one probeset
listed in FIG. 3A or 3B or 3C or 3D, wherein the presence of
KIR2DS4 in said donor is indicative for a reduced mortality, a
reduced graft-versus-host reaction, an increased graft-versus-tumor
reaction and an increased grafted organ survival in said recipient
as compared to the mortality, graft-versus-host reaction,
graft-versus-tumor reaction and grafted organ survival of a
recipient with a donor wherein KIR2DS4 is absent. In a preferred
embodiment probeset 504A/504B, and/or 708K/708L/708M/708N as
depicted in FIG. 3C and/or 3D are used for determining the presence
or absence of KIR polymorphisms.
[0086] A correlation has been established between the copy number
of KIR2DL2 and KIR2DS2 and rheumatoid arthritis (RA) with
extra-articular manifestations and rheumatoid vasculitis. A higher
copy number of KIR2DL2 and/or KIR2DS2 in an individual was
demonstrated to be predisposing for rheumatoid arthritis with
extra-articular manifestations and rheumatoid vasculitis (Majorczyk
et al 2007, Yen et al 2001). Additionally, rheumatoid arthritis
patients positive for KIR2DL3 and negative for KIR2DS3 had earlier
disease diagnosis (Majorczyk et al 2007).
[0087] The present invention provides probes that are particularly
well suitable for determining the presence or absence and copy
number variation of KIR genes, including KIR2DL2, KIR2DS2, KIR2DL3
and KIR2DS3. Thus, with probes according to the present invention
selected from FIG. 3A, 3B, 3C and/or 3D the presence or absence and
copy number of KIR2DL2, KIR2DS2, KIR2DL3 and KIR2DS3 in a sample is
particularly well determined. Preferably probe sets 420A/420B,
and/or 706A/706B and/or probe set 703A/703B/703C, and/or 544A/544B
as depicted in FIG. 3C and/or 3D are used to estimate the copy
number of KIR2DL2 and/or KIR2DS2. Preferably probe sets
415B/415C/415D and/or 417A/417B/417C and/or probe set 513B/513D
and/or 540A/540C as depicted in FIG. 3C and/or 3D are used to
estimate the copy number of KIR2DL3 and/or KIR2DS3. With probes
selected from FIG. 3 susceptibility of an individual to rheumatoid
arthritis (RA) with extra-articular manifestations and rheumatoid
vasculitis is thus particularly well determined.
[0088] Therefore in one embodiment the invention provides a method
for determining predisposition to rheumatoid arthritis with
extra-articular manifestations and rheumatoid vasculitis of an
individual comprising determining the copy number of KIR2DS2 and/or
KIR2DL2 in a nucleic acid sample of said individual with at least
one probeset listed in FIG. 3A, 3B, 3C and/or 3D, wherein a high
copy number of KIR2DS2 and/or KIRDL2 in said individual is
indicative for a predisposition for rheumatoid arthritis with
extra-articular manifestations and rheumatoid vasculitis as
compared with a low copy number of KIR2DL2 and/or KIR2DS2 in an
individual. In a preferred embodiment probeset 420A/420B, and/or
706A/706B and/or probe set 703A/703B/703C, and/or 544A/544B as
depicted in FIG. 3C and/or 3D are used for determining the copy
number of KIR genes.
[0089] Finally, a correlation has been found between the presence
or absence or copy number of KIR genes and predisposition to
autoinflammation, such as HLA-B27-related enthesitis-related
arthropathy and reactive arthritis, psoriasis, in individuals. For
instance, KIR3DL2 is increased in spondylarthritides and juvenile
enthesitis-related arthritis (Chan et al 2005, Brown 2009). The
present invention provides probes that are particularly well
suitable for determining the presence or absence and copy number
variation of KIR genes. Thus with probes selected from FIG. 3
susceptibility of an individual to autoinflammation, such as
HLA-B27-related enthesitis-related arthropathy and reactive
arthritis, psoriasis is particularly well determined.
[0090] Therefore, in one embodiment the invention provides a method
for determining predisposition to autoinflammation, preferably
HLA-B27-related enthesitis-related arthropathy and reactive
arthritis, psoriasis, in individuals comprising a) determining the
presence or absence and/or copy number of a KIR gene indicative for
said disorder in a nucleic acid sample of said individual with at
least one probeset listed in FIG. 3A or 3B or 3C or 3D, and b)
correlating the result obtained in step a) with presence or absence
of said predisposition.
[0091] In another embodiment the invention provides a method for
determining predisposition to spondylarthritides and/or juvenile
enthesitis-related arthritis of an individual comprising
determining the copy number of KIR3DL2 in a nucleic acid sample of
said individual with at least one probeset listed in FIG. 3A, 3B,
3C and/or 3D, wherein a high copy number of KIR3DL2 in said
individual is indicative for a predisposition for
spondylarthritides and/or juvenile enthesitis-related arthritis as
compared with a low copy number of KIR3DL2 in an individual. In a
preferred embodiment probeset 404A/404B, and/or 538A/538B/538D as
depicted in FIG. 3C and/or 3D are used for determining the copy
number of KIR genes.
[0092] The invention is further explained in the following
examples. These examples do not limit the scope of the invention,
but merely serve to clarify the invention.
BRIEF DESCRIPTION OF THE DRAWINGS
[0093] FIG. 1. A) Schematic outline of a conventional MLPA
reaction. The figure is adapted from www.mpla.com.
[0094] FIG. 1B) illustrates the use of two ligation sites in one
probe set, to detect two SNP's at the same time with one probe set
(a Tri-Lig probe) on a specific target sequence. If the correct
SNP's are present at both ligation sites, the three probe parts
will become ligated together to result in one PCR product, as shown
at the bottom left. If an incorrect SNP is present on one or both
ligation sites, no PCR product will be formed, as shown at the
bottom right.
[0095] FIG. 1C) illustrates the use of two ligation sites in one
Tri-Lig probe, to detect one particular gene, KIR3DL1*024N, in the
background of all other KIR3DL1 WT alleles at the first ligation
site, and all other KIR genes at the second ligation site. The 1a
probe detects all WT KIR3DL1 alleles (1a) whereas the 1b probe only
detects the KIR3DL1*024N allele (1b), due to a different SNP at the
first ligation site. The partial KIR gene sequences 2 to 12 are not
detected by the 1a and 1b probes, because these probes are only
specific for KIR3DL1 genes at the second ligation site due to a
different SNP at the second ligation site.
[0096] FIG. 2. IUB nucleotide codes of degenerate bases
[0097] FIG. 3 KIR-specific probe sets. A) KIR probe mix 1. Bold
nucleotides represent probes that are part of a probe set
consisting of three probes used for detection of two SNPs, B) KIR
probe mix 2. Bold nucleotides represent probes that are part of a
probeset consisting of three probes used for detection of two SNPs,
C) extended KIR probe mix 1. Bold nucleotides represent primer
binding sites. KIR genes in which two SNPs are detected using one
probe set according to the invention, consisting of three probes
are depicted in FIG. 13, D) extended KIR probe mix 2. Bold
nucleotides represent primer binding sites. KIR genes in which two
SNPs are detected using one probe set of this probe mix, consisting
of three probes are depicted in FIG. 13, E) control probe mix.
[0098] FIG. 4. The KIR protein structures. Depicted as large ovals
are the extracellular Ig-like domains, as squares the ITIMs and as
small light grey circles the charged residues on the cytoplasmic
tail (IPD KIRdatabase). Inhibitory KIRs and activating KIRs are
indicated by a "+" and "-", respectively.
[0099] FIG. 5. Exon structure of KIR3DL1. Exons are depicted with
black boxes and introns with lines and are draw approximately to
scale (Vilches et al, 2002).
[0100] FIG. 6. The organization of KIR locus. a: Framework genes
KIR3DL3, KIR2DL4 and KIR3DL2 are in black and are found at the
beginning, near the middle and at the end of the locus. The
pseudogenes KIR2DP1 and KIR3DP1 (which is also a framework gene) in
white and black, respectively, and the regions between the
framework genes are variable and these KIR genes are in grey, with
activating KIRs with black letters and inhibitory KIRs in white. b:
One example of haplotype A. c: An example of haplotype B (Parham et
al, 2003).
[0101] FIG. 7. The pedigrees of 12 families from the KIR reference
panel I (the families 1347 and 1349 are depicted in FIGS. 11 and
12, respectively). The four numbers on top of the pedigree is the
CEPH family number and the numbers in the shapes is the individual
number, these numbers correspond with the numbers in table 4. The
letters below the shape indicates the haplotypes and can be found
in the legend next to the pedigree.
[0102] FIG. 8. Electropherogram of probe set 1. The peak patterns
of the probes on two donors: 8080 (top) and 5911 (bottom). All 17
KIR probe peaks are present on donor 8080 and 10 KIR probe peaks on
donor 5911. In all donors the nine control probes (Ctr2-10) and the
probes on the four framework genes: KIR3DL3, KIR3DP1, KIR3DL2, and
KIR2DL4 (indicated with the black arrows) generated a signal.
Electropherogram of probes set 2 were similar for these two probe
groups (data not shown).
[0103] FIG. 9. Comparison of peak intensities of the probe 2DS2
(black arrows) between a true positive for KIR2DS2 (top) and a
false positive (bottom).
[0104] FIG. 10. The peak profiles of the probes 2DL5 (left arrows)
and 2DL5A (right arrows). Top: a sample which is positive for
KIR2DL5 indicated by the presence of the peak from probe 2DL5 and
the peak from 2DL5A cannot be distinguished in the presence of
KIR2DL5A or 3DP1*004. Bottom: this sample is negative for KIR2DL5
indicated by the absence of the probe 2DL5 and the peak of 2DL5A
indicates the presence of KIR3DP1*004.
[0105] FIG. 11. The pedigree of family 1347.
[0106] A) Left: The numbers of the individuals in top left pedigree
correspond with the numbers of the DNA samples in the table. At the
bottom the haplotype is denoted in letters and the legend for the
haplotype is displayed below (www.ihwg.org). The CNV of some of the
genes where quantified different by each of the two probe sets, the
number before `/` is for probe set 1 and after for probe set 2.
[0107] B1) Interpretation based on SSP-PCR data from CEPH-IHWG and
the conventional KIR haplotype model (see also
http://www.ncbi.nlm.nih.gov/projects/gv/mhc/xslcgi.fcgi?id=1347&cmd=kirpe-
d&locus_group=1).
[0108] B2) Novel haplotype model based on SSP-PCR data obtained
from CEPH-IHWG
(http://www.ncbi.nlm.nih.gov/projects/gv/mhc/xslcgi.fcgi?id=134-
7&cmd=kirped&locus_group=1).
[0109] B3) Copy number variation of KIR genes, determined using
SSP-PCR data obtained from CEPH-IHWG based on the conventional KIR
haplotype model (table 1) and the novel KIR haplotype model (table
2) and copy number variation of KIR genes, determined by KIR-MLPA
using the extended probe sets 1 and 2 and the novel KIR haplotype
model (table 3).
[0110] FIG. 12. The pedigree of family 1349.
[0111] A) Left: The numbers of the individuals in top left pedigree
correspond with the numbers of the DNA samples in the table. At the
bottom the haplotype is denoted in letters and the legend for the
haplotype is displayed below (www.ihwg.org). The CNV of some of the
genes where quantified different by each of the two probe sets, the
number before `/` is for probe set 1 and after for probe set 2.
[0112] B1) Interpretation based on SSP-PCR data from CEPH-IHWG and
the conventional KIR haplotype model (see also
http://www.ncbi.nlm.nih.gov/projects/gv/mhc/xslcgi.fcgi?id=1347&cmd=kirpe-
d&locus_group=1).
[0113] B2) Novel haplotype model based on SSP-PCR data obtained
from CEPH-IHWG
(http://www.ncbi.nlm.nih.gov/projects/gv/mhc/xslcgi.fcgi?id=134-
7&cmd=kirped&locus_group=1).
[0114] B3) Copy number variation of KIR genes, determined using
SSP-PCR data obtained from CEPH-IHWG based on the conventional KIR
haplotype model (table 1) and the novel KIR haplotype model (table
2) and copy number variation of KIR genes, determined by KIR-MLPA
using the extended probe sets 1 and 2 and the novel KIR haplotype
model (table 3).
[0115] FIG. 13. Detection of KIR alleles and KIR copy number
variation.
EXAMPLES
Example 1
[0116] This Example presents a new method for KIR genotyping.
[0117] KIRs are expressed by natural killer (NK) cells and a subset
of T cells. NK cells are cells of the lymphoid lineage, but display
no antigen-specific receptors. Their main function is to monitor
host cells for the presence of MHC class I molecules and this is
important for e.g. distinguishing healthy cells from virus-infected
or tumors cells. A low expression of MHC class I molecules on host
cells, which may for instance occur during viral infections as a
result of virus-mediated down regulation to prevent presentation of
viral peptides to CD8 T cells, stimulate NK cells to launch
cytotoxic attack. This phenomenon is also known as the "missing
self" theory.
[0118] NK cells express a variety of receptors that mediate
interactions with MHC class I molecules, including members of the
KIRs and CD94/NKG receptor multigene families. Interaction between
MHC class I molecules and these receptors regulates NK cytotoxicity
generally through the generation of inhibitory signals. The
composition between KIR and CD94/NKG families of humans and mice
differs considerably, with KIRs constituting the most in genetic
and gene number variation in man.
[0119] KIRs were first discovered in their role in fighting virus
infections by natural killer cells, but they are also expressed by
a subset of T cells. The KIR gene cluster is located at chromosome
19q13.4 within the leukocyte receptor complex (LCR) and spans a
region of about 150 kb. Up to 15 genes plus two pseudogenes have
been identified to date. Characteristic of the KIR gene cluster is
the variable gene content and an extensive degree of allelic gene
variants. The gene content between unrelated individuals can differ
considerably in the amount of KIR (pseudo)genes present, but also
in the numbers of activating and inhibitory (pseudo)genes.
Contractions and expansions by non-reciprocal recombination are the
major mechanism behind KIR diversification. KIRs can be divided
into two haplotypes, A and B in which haplotype B has a greater
variety in gene content and contains more activating KIR genes.
Studies of different ethnic populations show significant
differences in the distribution of these two haplotypes. The
selective pressures, such as exposure to different pathogens and
rapidly evolving MHC class I molecules appear to be the forces
behind such a gene diversification. A functional analog is the Ly49
gene family in mice, but KIRs and Ly49 are structurally distinct
proteins. KIRs have been identified in different primate species,
but they are species-specific and differ in gene content among
various species. These findings provide evidence for a rapid
evolution and expansion of this gene family.
[0120] Another level of relevant variation is the level of
expression of KIRs by individual NK cells. Each NK cell expresses
only a subset of its KIR gene repertoire and the presence of HLA
ligands seems to influence the frequency of NK cells expressing the
cognate ligand. A higher frequency of NK cells expressing
inhibitory KIRs in individuals have been found, when their cognate
HLA ligand is present. The ligands of some KIRs, in particular
those with activating potential remain to be determined.
[0121] Some of these activating KIRs seem to have lower affinity
for their cognate HLA class I ligands in comparison with their
related inhibitory receptors.
[0122] KIRs have been associated with several diseases, but due to
the genetic diversity between and in populations and the
differences in KIR expression by NK cells, a clear understanding of
their role has yet to be defined. KIRs have been reported to play a
role in allogeneic hematopoietic stem cell transplantation (HSCT),
which is used in the treatment of leukemia. It was suggested that
an intentional mismatch between donor KIR and recipient HLA ligands
would allow for a graft anti-tumor effect. KIR3DS1 and KIR3DL1 have
been reported to be associated with slower progression to AIDS and
several other virus infections, such as Hepatitis C virus (HCV),
human cytomegalovirus (CMV). Also the protozoan infection with
Plasmodium falciparum implicated roles for KIRs in malaria. In
autoimmune and inflammatory conditions, certain KIRs and cognate
ligand potentially results in higher susceptibility or protection
of the host.
The KIR Gene Cluster
[0123] The KIR acronym originally stood for killer cell-inhibitory
receptor, because the first KIR discovered had an inhibiting effect
on NK cells. To date, KIR is an abbreviation for Killer-cell
Immunoglobulin-like Receptor, as this family includes both
inhibitory and activating receptors. The HUGO Genome Nomenclature
Committee (HGNC) is responsible for the naming of KIR genes.
Currently KIR gene family consists of 15 genes and 2 pseudogenes,
listed in Table 1 (Marsh et al, 2002). KIR genes are named after
the protein structure they encode. The "D" denotes "Domain" and the
number 2 or 3 before it indicates the number of extracellular
Ig-like domains. "L" indicates a "Long" cytoplasmic tail and "S"
indicates a "Short" cytoplasmic tail and the "P" indicates a
"pseudogene". The number behind the letter L or S denotes the gene
encoding for this structure. Thus KIR2DL1 encodes for a structure
with two Ig-like domains and a long cytoplasmic tail. KIR2DL5A and
KIR2DL5B are exceptions; they were initially identified as one gene
KIR2DL5. However these two structurally similar variants are
discovered to be located on different regions of the KIR gene
cluster and can be inherited separately (Gomez-Lozano et al,
2002).
[0124] The KIRs that possess long cytoplasmic tails transduce
inhibitory signals to the NK cell, owing to the two immunoreceptor
tyrosine-based inhibitory motifs (ITIMs) (FIG. 4). Binding of these
receptors with HLA class I molecules leads to phosphorylation of
the tyrosine residues within the ITIM. Tyrosine phosphatase (SHP-1)
is then recruited and activated by the ITIM and prevents or
inhibits phosphorylation events which are associated with cellular
activation. NK-cell mediated cytotoxicity and cytokine secretion
inhibition are the main downstream effects. Short cytoplasmic tails
lack the ITIM and possess a basic charged amino acid, such as
lysine in the transmembrane domain. This positively charged amino
acid residue allows association with an adaptor molecule, such as
DAP12. DAP12 has one immunoreceptor tyrosine-based activation motif
(ITAM). When the tyrosine residues in the ITAM are phosphorylated a
docking site for SH2 domain of ZAP70 and Syk tyrosine kinase is
generated. The action of these kinases triggers a downstream
transduction cascade that promotes NK-mediated cytolysis (Middleton
et al, 2005). KIR2DL4 is unique among KIRs, as it possesses a long
cytoplasmic tail with a charged amino acid arginine in the
transmembrane region. KIR2DL4 might therefore be capable of
eliciting both activating as well as inhibitory signals.
Exon and Intron Structure
[0125] The KIR3DL1 and KIR3DL2, with three extracellular Ig-like
domains represent the prototypical KIR from which all the others
can be derived. KIR genes are organized in nine exons, the order of
these exons corresponding to the different functional regions of
the protein (FIG. 5). The first two exons encode the signal
peptide, exons 3, 4 and 5 encode the Ig-like domain, D0, D1 and D2,
respectively. Exon 6 encodes the stem or linker that connects the
D2 domain with the transmembrane region that is encoded by exon 7.
Exons 8 and 9 encode the cytoplasmic tail. Type 1 KIRs have two
Ig-like domains D1 and D2, KIR2DL1-3 and KIR2DS1-5. The protein
products of type 1 lack the D0 domain because exon 3 is a
pseudo-exon. This exon is spliced out of the RNA transcript,
possibly due to a three-base-pair deletion. Type 2 KIRs have the D0
and D2 domains, KIR2DL4-5, exon 4 is absent in these KIR genes,
resulting in a protein without D1 domain.
[0126] In KIR2DP1 exon 3 is a pseudoexon and exon 4 has an early
stop codon. If KIR2DP1 would be transcribed this could result in a
KIR protein with only a single Ig (D2) domain. In KIR3DP1 exon 2 is
missing due to a deletion. The exons encoding for the stalk, TM and
cytoplasmic regions are also absent. The three exons coding for the
Ig-like domains are intact, however the leader sequence is missing.
No transcripts have been found for KIR2DP1 (Trowsdale et al, 2001)
and KIR3DP1, the latest one is normally silent, but a recombination
of KIR2DL5A and KIR3DP1 have been found to be transcribed and is
predicted to be secreted rather than anchored to the cell membrane
(Gomez-Lozano, 2005).
Genotypes
[0127] Uhrberg et al. (Uhrberg et al, 1997) identified that the KIR
locus in humans appeared to be polygenic and polymorphic.
Individuals have a variable KIR gene content, achieved through
differences in number of total KIR genes and differences in the
amount of activating and inhibitory KIR genes. The mechanism behind
the KIR diversification is non-reciprocal recombinations between
non-allelic genes leading to expansion and contractions of the KIR
locus. Also reciprocal crossing over events are postulated to
contribute to the diversity. The KIR locus can be separated into
two parts with KIR3DL3 on the centromeric end and the central
KIR3DP1 on one half, and KIR2DL4 in the central and KIR3DL2 on the
telomeric end on the other half. Inside these two parts of KIR
locus, genes are located that are in much stronger linkage
disequilibrium, supporting a homologous recombination event
(Uhrberg 2005).
[0128] Studies worldwide using genomic DNA to determine the
presence or absence of KIR genes in populations have contributed to
an extensive amount of KIR-genotype profiling data. These studies
show a difference in frequency of KIR genes in populations of
different ethnic backgrounds and can be found on
www.allelefrequencies.net. The methods used for KIR genotyping are
polymerase chain reaction with sequence-specific primers (PCR-SSP),
sequence-specific oligonucleotide probes, PCR (PCR-SSOP), multiplex
PCR, automated sequencing and mass spectrometry.
Haplotypes
[0129] KIR genes can be divided in the haplotypes A and B
(Carrington et al, 2003). Both haplotypes contain the framework
genes KIR3DL3, KIR3DP1, KIR2DL4 and KIR3DL2. These genes are
conserved and are virtually present in every individual. Haplotype
A is uniform in terms of gene content and is composed of five
inhibitory genes (KIR3DL3, KIR2DL3, KIR2DL1, KIR2DL4KIR3DL1 and
KIR3DL2, and only one activating KIR2DS4, as shown in FIG. 6.
However the central framework gene KIR2DL4 may have an activating
function. On the other hand, there are haplotypes A that possess
null variants of both KIR2DS4 and KIR2DL4 that are not expressed on
the cell surface and technically these haplotypes contain virtually
no functional activating KIR.
[0130] Haplotype B is more variable than haplotype A and is
characterized by one or more of the following genes: KIR2DS2,
KIR2DL2, KIR2DL5, KIR2DS3, KIR3DS1, KIR2DL5A, KIR2DS5 and KIR2DS1,
conversely haplotype A is characterized by the absence of these
genes. The frequency of both haplotypes is relatively even among
populations of different ethnic background. It is possible that
some haplotypes cannot be placed in these two categories, as the
definition of haplotypes varies between authors and hybrids of
haplotypes are possible (Vilches et al, 2002). Distinction between
A and B haplotypes is useful in biological and medical settings, as
haplotype B have more genes that encode for activating KIR than
haplotype A. The haplotypes have been constructed by family
segregation analysis, genomic sequencing and gene-order analysis
(Shilling et al, 2002). FIG. 6 depicts the organization of a KIR
locus.
Gene Variation
[0131] Adding another level of genetic diversity to the KIR family
is the extensive degree of gene variations, which are exhibited by
all KIR genes. Allelic diversity is generated by substitutions of
nucleotides, recombination or gene conversion and point mutations.
Activating KIRs and inhibitory KIRs share a high sequence homology.
Activating KIRs are believed to be derived from inhibitory KIRs by
alterations in sequence, creating a charged residue upstream of a
stop codon and an elimination of ITIMs. Due to their younger
evolution, allelic diversity of activating KIRs is quite limited
when compared to inhibitory KIRs, but the variation of activating
receptors across ethnic populations is more extensive.
[0132] Currently a total of 335 KIR alleles have been identified
and can be found at the website: http://www.ebi.ac.uk/ipd/kir
(table 2). KIR allele sequences are denoted by an asterisk after
the gene name. Differences in the encoded protein sequences are
distinguished by the first three digits, the next two digits are
used to denote alleles that differ by synonymous differences within
the coding sequence (i.e. not resulting in amino acid
substitutions) and the last two digits are used for alleles that
have differences in the noncoding region, such as introns and
promoters. Thus, 3DL1*009 and 3DL1*010 are alleles that encode
different protein products and 3DL1*00101 and 3DL1*00102 are
alleles that encode the same protein product, but these alleles
differ by a synonymous DNA substitution within the coding region
(Marsh et al, 2002).
Expression and HLA
[0133] The ligands for inhibitory KIRs are MHC class I molecules,
which are constitutively expressed by most healthy cells, but can
be down-regulated in tumors and infected cells allowing killing by
NK cells. Interaction of MHC with inhibitory receptors ensures
tolerance of NK cells towards self. MHC class I molecules are
encoded by human leukocyte antigen (HLA) genes that are located at
chromosome 6p21.3 and are polymorphic and display significant
variations. KIR genes and HLA genes segregate independently during
meiosis, because they are located on different chromosomes. This
can lead to interesting HLA and KIR combinations inherited by one
individual, but to obtain a functional interaction between receptor
and the cognate ligand, they need to be expressed together. This
raises the question whether a correlation exists between the genes
encoding KIR and HLA. The ligand specificity for activating KIRs is
not well defined. The ligands of some activating KIRs have not been
identified yet. The activating receptors of KIR2DS2 and KIR2DS1
were reported to have a lower affinity of binding to HLA-C than
those of their closely related inhibitory receptors. It is also
possible that non-HLA ligands exist for these activating KIRs. The
KIRs with a defined cognate ligand are presented in table 3.
[0134] The KIR surface protein repertoire in an individual is
mainly determined by the KIR genes. Hence, a lack of expression is
more likely caused by the lack of that gene than by a
down-regulation. KIR genes are expressed by NK cells in a clonal
manner, each individual NK cell within a person possesses a
different combination of KIRs, with a subset of the total KIR gene
repertoire being expressed on each individual. KIR2DL4 is one
notable exception; this gene is ubiquitously expressed on NK cells.
The frequency of each expressed KIR may differ between individuals,
but is stable over time. For example the gene KIR2DL1 may be
expressed on 50% of the NK cell population of individual A, while
in individual B the expression of KIR2DL1 is found to be 14% of its
NK cell population. One explanation for this difference could be
that particular alleles of a gene are expressed more frequently due
to the presence of multiple copies of a gene.
[0135] This Example presents a new method for KIR genotyping with
multiplex ligation dependent probe amplification (MLPA). With this
method a rapid and convenient way of KIR genotyping is performed
and also the relative number of copies of the KIR genes is
quantified. Copy number variation (CNV) accounts for a substantial
amount of genetic variation, resulting in significant phenotypic
variations in e.g. transcript levels and therefore are of
functional relevance.
[0136] We developed two synthetic MLPA probe sets for the typing of
16 out of the 17 KIR genes KIR2DL1-5, KIR2DS1-5, KIR3DL1-3,
KIR3DS1, KIR3DP1 and KIR2DP1. The probes for the KIR genes were
designed for different loci to detect most of the alleles.
Probesets 1 and 2 are listed in FIGS. 3A and 3B. The specificity of
the probes was validated by comparison of the samples for the KIR
genotypes obtained with PCR-SSOP and PCR-SSP methods, and the
ability of the probes to quantify relative gene copy numbers was
examined with 12 families, each consisting of two parents and two
offspring, which have been genotyped for most KIR alleles.
Materials & Methods
DNA Selection/Isolation
[0137] DNA from unrelated randomly selected Caucasian donors was
obtained for this study to test the peak profile of the probes. For
the validation of the probes five SSP-PCR KIR typed genomic DNA
samples and 11 EBV transformed B cell lines from the 10.sup.th
International Histocompatibility Workshop were used (Cook et al,
2003), JVM, T7507, OLGA, SAVC, JBUSH, BM16, LBUF, AMALA, BM90,
TAB089 and KAS116. The KIR Reference Panel I from the IHWG
containing 48 samples from 12 Centre de'Etude du Polymorphism
Humain (CEPH) families--including 2 parents and 2 children (table
4: KIR typing of the 48 samples and FIG. 7: the pedigrees)--also
served this purpose, but its main purpose was to determine the
ability of copy number quantification of the probes. Genomic DNA
and the DNA from the Cell lines were isolated with Qiagen (blood
kit) according to the manufacturer's instructions.
Probe Design
[0138] Probes were designed according to general instructions
(www.mlpa.com/protocols.htm). All the probes were manufactured by
Invitrogen (Carsblad, Calif.). The sizes of the probes after
ligation ("ligated probes") are spaced four to five nucleotides
apart, to separate each amplification product on the sequence type
gels, amplification product size ranged from 95 to 223 nucleotides.
All MLPA probes contain a PCR primer sequence, which is recognized
by a universal primer pair. PCR primer sequences were: forward
5'-GGGTTCCCTAAGGGTTGG-3' and reverse
5'-TCTAGATTGGATCTTGCTGGCAC-3'.
[0139] The KIR probes were designed to identify and discriminate
between the 17 KIR genes listed in table 1, with exception of
KIR2DL5B. No specific probe could be designed for this gene. The
probe for KIR2DL5 now, detects both KIR2DL5A and KIR2DL5B genes. In
addition probes on alternative sequences and intron sequences were
designed, using basic local alignment sequence tool searches and
the IPD/KIR Database, http://www.ebi.ac.uk/ipd/kir. The sizes of
the KIR probes can be found in tables 5 and 6.
[0140] The targets of the nine control probes are on conserved
genes in the human genome, FGF3, BCAS4, LMNA, PARK2, MSH6, GALT,
SPG4, IL-4 and NF2. These target genes were tested to show no
considerable variation between donors in a previous MLPA study at
Sanquin. Control 1 and 10 were initially 88 bp and 130 bp
respectively, but have been elongated to 180 bp and 223 bp to
distribute the control probes more evenly among the KIR probes.
Table 7 shows the list of the genes and the sizes of the control
probes.
[0141] Competitor probes are designed where the signal of the probe
was off-scale to be detected by the capillary electrophoresis
apparatus and are listed in table 8.
MLPA Reaction
[0142] All DNA samples were diluted to 20 ng/.mu.l with water and 5
.mu.l was denatured at 98.degree. C. for 5 minutes in 200 .mu.l
tubes in a Biometra T-1 Thermoblock with heated lid.
[0143] MLPA reagents (EK kit 5) were obtained from MRC-Holland
(Amsterdam, The Netherlands). SALSA MLPA buffer (2 .mu.l) and 1-10
fmol of each MLPA probe in a probe mixture (1 .mu.l) were added and
incubated for 1 minute 95.degree. C., followed by 16 hours at
60.degree. C. in a total volume of 10 .mu.l. Ligation of the
hybridized probes was performed by reducing the temperature to
54.degree. C., before adding 32 .mu.l Ligase-65 mix (3 .mu.l ligase
buffer A, ligase buffer B, 1 .mu.l Ligase-65 and 25 .mu.l water)
and incubated for 15 min. After inactivating the enzyme at
98.degree. C. for 5 min, 10 .mu.l of the ligase mix was diluted
with 4 .mu.l PCR Buffer and 26 .mu.l water at 4.degree. C. in 200
.mu.l tubes. For the PCR reaction, 10 .mu.l of polymerase mix (0.5
.mu.l polymerase, 2 .mu.l SALSA enzyme dilution buffer, 2 .mu.l
SALSA PCR-primers and 5.5 .mu.l water) was added at 60.degree. C.
PCR amplification of the ligated MLPA probes was performed for 36
cycles (30 sec 95.degree. C., 30 sec 60.degree. C., 60 sec
72.degree. C.) followed by an incubation for 20 min at 72.degree.
C.
Electrophoresis
[0144] 1 .mu.l PCR product is added in new tubes containing 0.4
.mu.l Promega Rox size standard 60-400 bp+8.6 .mu.l High Definition
buffer. The products are separated by Applied Biostystems Genetic
Analyzer 3130XL capillary electrophoresis according to its
molecular weight and the resulting electropherogram show specific
peaks that correspond to each probe.
Analysis
[0145] Data were visualized with Genemapper v3.6 and normalized
with Soft genetics Genemarker v1.6, using internal control probe
normalization (http://www.softgenetics.com/papers/MLPA). Finally
these data was exported to an Excel file.
Results
Detection of Probe Signal
[0146] All the MLPA probes were initially tested on randomly chosen
donors. We first examined if the probes would generate a signal and
if these signals corresponded with the expected size of each probe.
The control probe peaks and the probe peaks for the four framework
genes, KIR2DL4, KIR2DL3, KIR3DL3 and KIR3DP1, occurred in all
samples, as expected. KIR gene content variation between
individuals was observed when different samples were compared, FIG.
8. The probe intensity is denoted by arbitrary units (AU) on the
y-axis and the probe size is expressed on the x-axis in basepairs
(bp). We used the peak height to quantify the data, while others
may suggest probe area.
[0147] Secondly, the intensity of the probe signal was examined.
The peak patterns were visualized with Genemapper, to observe the
peak intensities before normalization. Genemarker is used to
normalize the data and correct this for the decay of larger probes,
but does not indicate where signals are off-scale. It is preferred
to have a probe signal between 500-6000 AU in order to obtain a
more reliable DQ value. Moreover fluorescent peaks with a signal
less than 500 AU may not always be detected when more probes are
added to the reaction. Fluorescent peaks above 6000 AU can be
off-scale to be detected by the sequencer and decrease the signal
of other probes relatively. Several suggestions are described to
enhance or lower probe intensity, the nucleotide composition next
to the PCR primer tag sites and/or the GC content of a probe are a
few factors that can be of influence (www.mlpa.com/protocols.htm).
In general competitors are used for reduction of probe signals and
a higher probe concentration for an increase in signal. Competitors
are oligonucleotides that are identical to a part of the MLPA probe
without the forward or reverse primer sequence, depending whether
the left or right part is chosen.
[0148] Competitors compete with the MLPA probe for the same target,
however no amplification of these ligated probes will occur, since
they lack a primer sequence. The result is that less probe
amplification product will be detected and lower peak intensity is
obtained.
[0149] Competitors were designed for control probes 2, 3, 4, 7 and
9 and in the first place also for the KIR probes 2DL4, 3DL3 (probe
set 1) and 3DL2 (probe set 2) These probes had a length of 96 bp,
100 by and 108 bp, respectively. However we observed a decrease in
peak intensity, more or less corresponding with an increase in
probe size. Longer synthetic probes are more likely to contain a
higher proportion of incomplete oligonucleotides. Therefore it
seemed to be an option to elongate the length of probes with high
peak intensities and to shorten this for probes with low peak
intensities. Probe 2DL4 was redesigned to 170 bp and 3DL3 to 154 bp
and lower peak intensities were the result. The peak generated by
probe 3DL3 (100 bp) was not affected by its competitor and was
apparently a product of the probe 2DS3 (108 bp), because when this
probe was removed from the probe set 1, the off-scale signal
reduced to normal. Furthermore competitors with a length of 30 bp
had less effect than those with a length of 50 bp, in which case a
higher dosage was needed to reduce the probe signal (data not
shown).
[0150] For probes that failed to generate a signal or for which the
signal was insufficient, the followings have been performed; a
three- to ten-fold concentration of these probes was used and
probes that have a high overlap in sequence were not included in
one probe set. Placing two cytosine nucleotides after the forward
primer should increase the probes signal and a tyrosine base should
decrease this, reported in the MLPA design protocol. However in our
experiment, several probes were redesigned to contain two cytosines
after the forward primer and this did not produce the same results.
Probes that still failed to generate a signal after the
aforementioned proceedings and testing on lager number of donors
were replaced by probes on the reverse strand of the target gene or
by probes that have a different target location on that gene.
[0151] The frequencies of each KIR gene probe peak on the tested
samples were compared with the KIR gene frequencies in Caucasian
population available on www.allelefrequencies.net (table 9). Probes
with observed frequencies that were contradicted by the population
frequencies were assumed to give false negative or false positive
results and were replaced by new designs. These were assumed to be
caused by gene variation at the ligation sites of the probe.
[0152] The list of the alleles that can be detected by the KIR
probes and the coverage of the total KIR alleles by the probes are
shown in table 10.
Other Factors Interfering with Peak Intensities
Probe Quality
[0153] We experienced differences probe quality by probes that were
manufactured at different companies. The nine control probes were
initially ordered from Biolegio (www.biolegio.com) which had also
supplied these for the C4 MLPA project previously done here. All
the KIR MLPA probes were ordered at Invitrogen
(www.invitrogen.com). The control probe set was separated in two
mixes, control probes 1 (IL-4), 2 (FGF3), 3 (BCAS4), 4 (LMNA), 5
(PARK2) and 7 (MSH6) in one and the control probes 8 (GALT), 9
(SPG4) and Ctrl 10 (NF2) in the other. The concentration needed for
each control probe varied and ranged from 0.5 fmol to 6 fmol and
also different concentrations of competitors were needed.
[0154] The control probes used for the KIR MLPA were ordered from
Invitrogen. Only 1 fmol is needed for each control, with the
exception of control probe 5 (3 fmol) in order to obtain the same
peak intensity as mention above and the probes do not need to be
separated into two mixes. Due to the better probe quality, time is
saved in producing the probe sets.
Template DNA Amount
[0155] A MLPA reaction with 50 ng of DNA was performed and compared
with 100 ng that is used throughout this study. MLPA reactions
using a DNA amount of 20 ng have been reported by Schouten et al.
(Schouten et al, 2002). When the peak profiles were compared, no
striking differences between these two reactions were observed. The
DQ of the nine control probes were calculated for each sample and a
sample with 100 ng DNA was taken as reference. Seven out of eight
samples containing 50 ng of DNA showed a DQ value outside [0.8-1.2]
for more than three control probes, ranging from [0.3-1.5] within
one sample. While all the eight samples of 100 ng DNA had DQ within
the acceptable range [0.8-1.2] for all the nine control probes,
with exception of one sample that had two control probe DQ value
outside this range. Here we conclude that MLPA reactions with
different amounts of DNA cannot be compared with each other,
because the DQ values of the same sample did not yield the same
score with the different DNA amounts.
[0156] Next the samples of 50 ng of DNA were compared among, by
taking a sample of 50 ng DNA as reference. The observation was that
three of the eight samples had more than three control probes with
a DQ value out of the range of [0.8-1.2]. When the nine control DQ
values of one sample were analyzed, values between [0.5-1.7] were
found. Therefore MLPA reactions carried out with 50 ng of DNA were
considered to be unreliable, as the DQ values of the probes showed
a great variation between the samples and within one sample, which
was not observed with the samples that contained 100 ng of DNA. The
requirement of higher amounts of DNA for this study could be
explained by the fact that we are using a completely synthetic
probe set in contrast with the probe sets used by Schouten et al
(Schouten et al, 2002). Moreover most studies that were carried out
with little amount of DNA often only analyzed chromosomal
abnormalities, such as recombination or mutations and did not
quantify copy numbers.
Reproducibility
[0157] Samples of different runs were not always comparable, when
the DQ of the control probes were calculated. The explanation is
that the experimental conditions may vary with each run, due to
human acting or differences in probe signal reproducibility.
Therefore, samples within the same run are preferably normalized
and analyzed first before comparing the data with samples of a
different run. Reference samples with a more or less established
relative gene copy numbers, are preferably included in each
experiment to act as reference.
Validation with KIR Typed DNA Samples
[0158] The specificity of the KIR probes was verified by testing 11
EBV-transformed cell lines, which were KIR-genotyped by the
10.sup.th International Histocompatiblity Workshop (IHW) (Cook et
al, 2003). The cell lines were KIR-genotyped using PCR-SPP and
PCR-SSOP and were carried out in three separated laboratories. The
cell lines were not genotyped for the genes KIR2DL5A, KIR3DL3,
KIR2DP1 and KIR3DP1 and also contained no negative controls for the
genes KIR2DL1, KIR2DL4, KIR3DL1, KIR3DL2 and KIR2DS4.
[0159] In addition, DNA samples from 5 individuals were genotyped
by PCR-SSP for further verification. These 5 samples were also
genotyped for the genes KIR3DL3 and KIR3DP1 and found to contain
true negative genotypic results for KIR2DL1 and KIR2DP1. The
results of the verification of the two probe sets are shown in
tables 11-14.
Probe Set 1
[0160] KIR genotyping with probe set 1 was found to be consistent
with the 10.sup.th IHW on 10 of the cell lines for the probes
2DL1-5, 2DS1, 2DS3-5, 3DL1-2 and 3DS1. All cell lines were typed
positive for the genes KIR2DP1, KIR3DP1 and KIR3DL3, the first has
a frequency between 94-100% (table 9) and the last two are
framework genes that are always present. Typing of the 5
individuals yielded the same results as with the PCR-SSP, except
for the probe 2DS2.
Probes for 2DL5A (Same Probe in Probe Set 2)
[0161] Most studies on KIR genotyping detect the presence of
KIR2DL5 and do not differentiate this gene between the two genes
KIR2DL5A and KIR2DL5B. These two genes show a nucleotide sequence
difference of only 1%. We were unable to design a probe for
KIR2DL5B, because a specific ligation site to discriminate KIR2DL5B
from KIR2DL5A and the other KIR genes was not found. The probes
that were designed for KIR2DL5A also detect the allele KIR3DP1*004
(table 10), because this allele contains no other difference in the
sequence within the probe's range, thus the probe sets do not
contain specific probes for the selective detection of KIR2DL5A. In
fact, KIR3DP1*004 is non-expressed, and forms a hybrid of the
promoter of KIR2DL5A and the coding region of KIR3DP1. When probe
2DL5A generates a signal in the MLPA, this could indicate the
presence of both KIR2DL5A and KIR3DP1*004 or either 2KIRDL5A or
KIR3DP1*004 alone. However, probe 2DL5 detects the same KIR2DL5A
alleles as probe 2DL5A. When probe 2DL5 is not binding and probe
2DL5A is, the absence of KIR2DL5A and the presence of KIR3DP1*004
is demonstrated. This is clearly demonstrated by the cell lines
JVM, SAVC, JBUSH, BM16, TAB089, KAS116 and the individuals
33.sub.--8025 and 33.sub.--8588 (FIG. 10).
Probe Set 2
[0162] Probe set 2 contains a smaller proportion of probes. A
higher proportion of the probes had overlapping sequences and seven
out of the ten KIR probes needed a 10-fold higher concentration
than the others to obtain peak intensities above 500 AU.
Probe 2DS5 and 3DS1
[0163] Probes 2DS5 and 3DS1 bound to all samples including to those
genotyped negative for KIR2DS5 and KIR3DS1, indicating unspecific
ligation of the probes. Probes 2DL5 and 3DS1 were not based on
primer sequences used before, the probe search tool on the KIR
database and BLAST results showed no match with other KIR genes and
these probes were considered to be specific for KIR2DS5 and
KIR3DS1. No explanation could be found, why these probes gave false
positive results. These probes were excluded from probe set 2.
Probe 2DS1
[0164] Three out of the six negative cell lines for KIR2DS1 were
typed positive by this probe, while the two negatives from the
PCR-SSP-typed individuals were correctly typed. Probe 2DS1 target
is on an intron and only little information about intron sequences
is available. The fact that other KIR genes may possess the same
sequence at this position, cannot be excluded and therefore this
probe is not included in the probe set.
Probe 3DP1
[0165] The probe 3DP1 in probe set 2 detects a deletion of exon 2,
this allele of KIR3DP1 is designated as KIR3DP1*003 and has a
frequency of 0.72 in the Caucasian population. Sample 33.sub.--8588
of the PCR-SSP typed individuals was typed negative for KIR3DP1 bp
the MLPA probe and positive by PCR-SSP (table 14). The conflicting
typing results between these two methods can be explained by the
presence of exon 2 in this sample.
Cell Line LBUF
[0166] Both probe sets have genotyped this cell line positive for
KIR2DL3 and negative for KIR2DL5 and KIR2DS. In addition, probe set
1, typed LBUF negative for KIR2DS1, KIR2DS5 and KIR3DS1 (table 11
and 13). It is reasonable to assume that the cell line LBUF that
was tested, was not the same as published before by the 10.sup.th
IHW. LBUF had been KIR-genotyped by Hsu et al. 2002 (Hsu et al,
2002) and their typing was consistent with ours. Moreover, LBUF and
the other cell lines was KIR-genotyped with the standard PCR-SSP
method and these results confirmed our findings with MLPA,
including the positive typing results of the genes KIR3DL3, KIR2DP1
and KIR3DP1 on all 11 cell lines.
Quantification of Gene Copy Numbers
[0167] For the verification of gene copy number quantification,
samples with a well-defined number of copies of KIR genes were
needed. Since these are not available, we used the KIR reference
panel I for this purpose, comprising 12 families of two parents and
two children each. These 48 reference samples have been
KIR-genotyped by 15 different laboratory groups utilizing PCR-SSP
and PCR-SSOP. The Centre de'Etude du Polymorphism Humain (CEPH),
Foundation Jean Dausset, Paris, France (www.cephb.fr), had prepared
lymphoblastoid cell lines (LCLs) of these families. The
International Histocompatibility Working Group (IHWG) Cell and DNA
Bank has made this panel available for commercial use
(www.ihwg.org).
[0168] All the samples have been identified for the presence or
absence of 16 of the KIR genes and for two variants of KIR3DP1,
(KIR3DP1*003 and KIR3DP1v) and two variants of KIR2DS4 (KIR1D alias
KIR2DS4*003 and KIR2DS4) (table 4). Whereas, KIR3DP1 of the KIR
reference panel I is characterized by the absence of exon 2 and the
KIR3DP1v indicates the remaining KIR3DP1 alleles. KIR1D contains a
22-bp deletion in Ig-like domain D2, causing a frame shift and
early stop codon which lead to a truncated protein product (Hsu et
al, 2002).
[0169] The haplotypes of these six families were also available as
shown in FIG. 7. In addition this figure shows the pedigrees of the
12 families. Because of the information about the haplotypes, we
could assume that some samples exhibit at least two copies of KIR
genes. The inheritance patterns of these copy numbers was deduced
from the pedigree information. The reference panel has at the same
time been utilized as an extra verification step for the
specificity of the probes.
Specificity in KIR Genotyping
[0170] With both probe sets difficulties were experienced with
generating reliable data of the MLPA experiments with the KIR
reference panel, presumably this is caused by the lower quality of
the DNA samples, as this did not occur with the genomic DNA samples
of the previous experiments. The DQ values of the control probes
had a higher frequency outside the proposed normal range [0.8-1.2].
Therefore, data of a number of samples is missing and these samples
should be tested in the future.
Probe Set 1
[0171] 16 probes: 2DL1-5A, 2DS1, and 2DS3-5, 3DL1-3, 3DS1, 2DP1 and
3DP1 were tested and the majority of the probes genotyped the KIR
reference panel accordingly to what has been reported, except there
were some differences with probes 2DP1 and 2DL5. These samples were
correctly typed by probe set 2.
Probe Set 2
[0172] The probes: 2DL1-5A, 2DS2, 2DS4, 3DL1-3, 3DS1, 2DP1 and
3DP1, in total 14 probes were tested on the reference panel. Probe
3DP1 was designed for KIR3P1*003 (denoted as 3DP1 in table 4) and
its specificity for this allele was confirmed with the reference
panel. Probe 2DL2 typed approximately 58% false positive and probe
2DL1 typed three of the four negative of the panel to be positive
and, therefore, no further testing has been done with these two
probes. Probe 2DS2 typed around 15% incorrectly as negative,
although in a previous run which was rejected because of the DQ
values of the controls, these two samples were typed positive.
These samples need to be revised before a conclusion about probe
2DS2 can be drawn. Probe 2DS4 gave one false negative result
(sample 1333-8281). Only 80% of the KIR2DS4 alleles can be detected
by this probe because of a gene variant that is 4 bases away from
the ligation site in 1 out of 9 alleles. The right part of this
probe will be redesigned with an UIB code on this position.
Quantification of CNV
[0173] Probes that have been demonstrated to be accurate in KIR
genotyping in both probe sets have been analyzed for their ability
in copy number quantification. Relative quantification of CNV with
one probe is simply not reliable because gene variations near the
ligation site of the probe may influence the outcome in DQ value.
This is especially true for KIR sequences, because they show a high
level of gene variation, while demonstrating a homology up to 99%.
Certain probes discriminate the different KIR genes only by one
nucleotide difference at their ligation site. A gene variant near
the ligation site of the target gene may lead to a lower probe
signal. Alternatively, a gene variant at one of the other KIR genes
might cause a probe to recognize this gene as its target, thus
enhancing the probe signal. Therefore only the KIR genes of the
families with the reported haplotype and the complete MLPA data of
the two probes are analyzed for copy numbers.
[0174] The DQ values of the control probes of both probe sets on
each sample were compared to check if the MLPA data are reliable.
The nine control probes should generate the same DQ values as these
control probes are the same in both probe sets and are tested on
the same sample. Samples with less than seven comparable control
probe DQ values between the two probe sets were excluded. Next, the
DQ values of the KIR probes were evaluated. We interpreted the
following; DQ values of 0.3< as 0 copies of that gene, DQ
[0.4-0.7]=1 copy, DQ [0.8-1.2]=2 copies, DQ [1.3-1.7]=3 copies, DQ
[1.8-2.2]=4 copies, DQ [2.3-2.7]=5 copies, etc. The borderline
values, such as a DQ of 0.7 are questionable and when the second
probe obviously quantified 1 copy of this gene, 0.7 was considered
as 1 copy, the same approach is applied with other borderline
values.
[0175] FIGS. 11A and 12A show the pedigrees of the families 1347
and 1349, respectively and the legends for the haplotype are
displayed below. The copy numbers of the KIR genes are listed in
the FIGS. 11A and 12A next to the pedigrees.
[0176] A difference in the quantification of the exact copy numbers
was observed with the probes for KIR3DP1 in samples: 1347-8445,
1347-8436 and 1349-8398. Probe set 1 seems to detect more copies of
this gene than probe set 2, which is in agreement with their
design. Probe 3DP1 (1) detects all the KIR3DP1(v) alleles and probe
3DP1 (2) detects only KIR3DP1*003 denoted in the legend as 3DP1,
which exhibit the exon 2 deletion. The probes 2DL3 and 2DL4 in
probe set 1 detected fewer copies numbers than their counterparts
in probe set 2. Probe 2DL3 and probe 2DL4 might have problems with
the presence of gene variants at their target sequence, whereas
these probes in probe set 2 have no gene variants in the probe
target sequence and give a coverage of 100% (table 10). The probes
for KIR3DL1 quantified the members of family 1349 differently. The
probe in probe set 1 covers different alleles than the probe in
probe set 2, the coverage rate are 78% and 41% respectively due to
gene variants present at their target sequence more then 10 bases
away from the ligation site, that might influence the binding
efficiency and thereby the peakhights. Also here adding IUB codes
in the probe sequence will overcome the problem of
misinterpretation of copy number differences between
individuals.
[0177] Despite the differences in copy number quantification of a
number of probes, the overall inheritance pattern of the gene
copies was in agreement with the inheritance of the haplotypes. For
example the four framework genes KIR3DL3, KIR3DP1, KIR2DL4 and
KIR3DL2 were present in all samples and at least 2 copies of each
of these genes have been found. This indicates that these genes are
present in at least one copy at each allele and are inherited from
both parents. Examination of family 1347 revealed that the father,
haplotype a/b (sample 8440) has three copies of gene KIR2DL5 on one
allele, haplotype b and one on the other, haplotype a and has past
haplotype b, with the three copies to the child (sample 8436) and
the allele haplotype a, with one copy to the other child (sample
8412). For the family 1349, one copy of KIR2DS4 is believed to
reside on one allele, haplotype c and two on the other, haplotype d
of the mother (sample 8399). Because both children, haplotype b/c
and haplotype a/c (sample 8393 and 8636), respectively, inherited
the allele with two copies from their mother as they have both the
haplotype c and one child (sample 8636) inherited one copy of this
gene from its father, haplotype a. Also when the inheritance
patterns of the remaining copy numbers of genes were analyzed, no
inconsistency with the inheritance patterns of the haplotypes could
be found. The rest of the families with fully reported haplotypes
should be tested again to obtain complete data of all the members
within one family, before the inheritance patterns and copy numbers
can be analyzed.
Discussion
[0178] Before the present invention, the main problem in designing
synthetic MLPA probes for KIR genotyping was to design probes
specific enough for the target gene, but still sensitive enough to
detect most of the alleles present in the population. KIR genes
have very high level of homology (85-99%) in the sequences of both
exons and introns and show an extensive degree of gene
variation.
[0179] The MLPA is a good method, because it can discriminate
target sequences that only differ one nucleotide at the ligation
site. The present inventors designed synthetic MLPA probes
consisting of three probe parts which added a second ligation site,
so that an extra discrimination point was provided. In addition
these three-part probes made it possible to elongate the ligated
probe size, the longest probe tested in this study was 223 bp (Ctr
10). Due to the better quality of the probes and three-part probes,
the number of probes in a synthetic MLPA probe set according to the
invention is less restricted by the size of the ligated probes.
[0180] This study has demonstrated that the MLPA with two synthetic
probe sets is reliable in KIR genotyping, as these two probe sets
have been well validated by three independent approaches. The two
probes sets complement each other in the detection and coverage of
the KIR alleles, which yielded in no false negatives any more in
all the samples used for verification. Even after exclusion of the
probes that may have generated false positives from the probe sets,
all 16 KIR genes can still be consistently detected for their
presence or absence. This makes the MLPA methods used in this
Example in a qualitative sense comparable to the PCR-SSP and
PCR-SSOP methods. However time and work is saved with the performed
Example, as only two reactions are needed to generate a complete
KIR-genotype profile.
[0181] In summary, probe set 1 contains the probes 2DL1-5, 2DS1,
and 2DS3-5, 3DL1-3, 3DS1, 2DP1 and 3DP1, in total 15 probes. Probe
set 2 contains the probes 2DL3-5, 2DS2-4, 3DL1-3, 2DP1 and 3DP1, in
total 11 probes. Together these two probe sets are accurate for the
typing of 16 KIR genes and for quantifying relative copy numbers of
at least 9 KIR genes.
Example 2
[0182] This Example presents additional probes for KIR genotyping
and copy number variation analysis with multiplex ligation
dependent probe amplification (MLPA). Here, probes are presented
for all 17 KIR genes KIR2DL1-5, KIR2DS1-5, KIR3DL1-3, KIR3DS1,
KIR3DP1 and KIR2DP1, including KIR2DL5a and KIR2DL5b, KIR3DP1v and
several null alleles. The extended probesets 1 and 2 are listed in
FIGS. 3C and 3D, respectively. As in example 1, the specificity of
the probes was validated by comparison of the samples for the KIR
genotypes obtained with PCR-SSOP and PCR-SSP methods, and the
ability of the probes to quantify relative gene copy numbers was
examined with 12 families, each consisting of two parents and two
offspring, which have been genotyped for most KIR alleles.
Materials & Methods
[0183] For DNA selection/isolation, probe design, MLPA reaction,
electrophoresis and analysis according to materials & methods
of example 1 with the exception that no competitors were used and
data were normalized with Soft genetics Genemarker v1.85, using
internal control probe normalization
(http://www.softgenetics.com/papers/MLPA) and synthetic
references.
Results
Extended Probesets
[0184] With the extended probesets 1 and 2 all KIR genes and
several KIR gene variants were detected.
[0185] The extended probe set 1 depicted in FIG. 3C detects the
same genes as probe set 1 of example 1 but additional probes are
added and therefore additional KIR gene variants are now detected.
Additional probes that are added are 2DL5B, 2DL4N
(2DL4*007,008,009,011), 3DL1*024N.
[0186] The extended probe set 2 as depicted in FIG. 3D detects the
same genes as probe set 2 of example 1 but additional probes are
added and therefore additional KIR gene variants are now detected.
Additional probes that are added are 2DL5B, 3DS1*049N and 2DS4N
(2DS4*004, *006, *007,*008 and *009). KIR2DS4N is also called
KIR1D.
Probe 3DP1
[0187] The probe 3DP1 in extended probe set 2 detects a deletion of
exon 2, this allele of KIR3DP1 is designated as KIR3DP1*003,
KIR3DP1*005 or KIR3DP1*006.
Probes for 2DL5A and 2DL5B
[0188] With the extended probesets 1 and 2 KIR2DL5A and 2DL5B are
now also detected. The probes that were designed for KIR2DL5A and
KIR2DL5B also detect the alleles KIR3DP1 variants (table 10,
KIR3DP1v). When probe 2DL5A or 2DL5B generates a signal in the
MLPA, this could indicate the presence of both KIR2DL5A and
KIR3DP1v or KIR2DL5B and KIR3DP1v respectively. Alternatively, when
probe 2DL5A or 2DL5B generate a signal in the MLPA the presence of
either KIRDL5A or KIR3DP1v alone (with probe 2DL5A) or KIR2DL5B or
KIR3DP1v alone (with probe 2DL5B) is indicated. Thus with these
probes 2DL5A and 2DL5B more than one KIR gene is detected.
Therefore, these probes are not suitable to determine copy number
variation (see FIG. 13).
Copy Number Variation (CNV)
[0189] For all KIR alleles except KIR3DP1 variants (KIR3DP1v),
KIR2DL5A and 2DL5B copy number variation is determined with
extended probesets 1 and 2 (FIG. 13).
Quantification of CNV
[0190] A difference in the quantification of the exact copy numbers
as compared to example 1 was elaborated by studies with the
extended probesets. Optimization of the probe set initially used in
FIG. 11A, has now resulted in a 100%-perfect match with the
validated KIR data in the in example 1 genotyped pedigrees. None of
the MLPA probes gave a false-positive or false-negative signal in
the 10.sup.th ICW families tested as exemplified by the analysis of
families 1347 and 1349 (FIGS. 11B & 12B). Thus, both probe set
1 and/or 2 and extended probe sets 1 and/or 2 are suitable for
detection of KIR genes and for determination of relative copy
number variation, but extended probe sets 1 and/or 2, as depicted
in FIGS. 3C and 3D, are preferred.
Specificity and Quantification for KIR Haplotyping
[0191] From the MLPA data within pedigrees haplotyping can be
inferred. First of all, the framework genes KIR3DL3 and KIR3DP1 for
the first block in both haplotypes A and B (FIG. 6) and KIR2DL4 and
KIR3DL2 are present in a fixed copy number of 2 genes. However,
KIR3DP1 may be present as so-called KIR3DP1v variant (see also FIG.
7, grey boxes represent the framework KIR genes in both haplotypes
A and B). In case of haplotype B the presence of KIR genes may vary
widely (FIG. 6), making this haplotype an important contribution to
the variation within the KIR gene cluster.
[0192] In family 1347, we have deduced, using the extended
probesets, from the pedigree a correct and complete KIR haplotype
analysis (FIG. 11B). At the single gene level the MLPA results
offers insight into the patterns of inheritance. The sibs inherited
from their parents different KIR haplotypes, which--for
instance--resulted in the variation in KIR2DL5 gene content. Thus,
both sibs have 2 of these genes, containing 2 KIR2DL5 genes from
the father (who carries 4 KIR2DL5 genes in total) and one
null-haplotype from the mother. From the present data from the
literature or the current MLPA data, it cannot yet be distinguished
whether the two KIR2DL5 genes that both sibs have inherited, are
the same alleles, or whether the KIR2DL5 are located in the first
or second block of the so-called B haplotype (see also FIG. 6).
[0193] At the haplotype level, patterns of inheritance are deduced
for the remaining non-framework KIR genes in this pedigree, e.g.
KIR2DL3, KIR2DS2, KIR2DL2, KIR2DP1, and KIR2DL1 genes in the first
block of haplotype B, generally located in between the framework
genes KIR3DL3 and KIR3DP1 genes (see also FIG. 6).
[0194] In case of the first block of haplotype B, the results are
explained by the inheritance of a KIR2DL3-KIR2DP1-KIR2DL1 haplotype
from the father and the KIR2DS2-KIR2DL2-KIR2DP1-KIR2DL1 haplotypic
block from the mother. In case of the second block of haplotype B,
it is clear that the KIR3DS1-KIR2DS3-KIR2DS1 haplotype has been
inherited from the father and the KIR3DL1-KIR2DS4 from the mother.
Yet, one sib (8436) must have lost a KIR3DL1 gene according to our
MLPA analysis. Sib 8436 has the normal 3DL1 present in our MLPA,
though sib 8412 has inherited a 3DL1N variant gene in stead of the
normal 3DL1 gene. This is just by normal inheritance so not an
exception.
[0195] SSP-PCR can not discriminate between 3DL1 variants (also not
between 3DS1 variant genes nor 2DL4 variant genes).
[0196] At the haplotype level, patterns of inheritance are
similarly deduced for the pedigree of family 1349 (FIG. 12B). Apart
from the framework KIR genes in this pedigree, the non-framework
genes form the haplotype B that are inherited "en bloc".
[0197] In case of these two sibs, 1349-8393 and -8636, the KIR
variation can be well explained by inheriting different KIR
haplotypes from both parents.
[0198] With respect to the first block of haplotype B, the results
are explained by the inheritance of one of his two similar
KIR2DL3-KIR2DP1-KIR2DL1 alleles from the father and one from the
mother (while this female also carried a smaller KIR2DL3-KIR2DP1
haplotypic block).
[0199] In case of the second block of haplotype B, it is clear that
the father carries a KIR3DL1-KIR2DS4 combination on one allele and
a separate KIR2DS3-KIR2DS4-KIR2DS1 haplotypic on the other allele
that were differently inherited by the two sibs, whereas the mother
carries two identical KIR3DL1-KIR2DS4 alleles.
[0200] In FIGS. 11 and 12 the standard SSP PCR results are compared
with our MLPA data with the extended probe sets 1 & 2 for the
pedigrees in the CEPH families 1347 and 1349.
[0201] Two KIR haplotype models have been described (see for
instance: H. Li, PLoS Genetics, 2008, 4, 11:e1000254; M. Uhrberg,
Eur. J. Imm. Highlights, 2005, 35:10-15; M. Carington, The KIR Gene
Cluster, 2003; K. Hsu, Imm. Reviews, 2002, 190:40-52). The
conventional KIR haplotype model assumes that there are two
haplotypes A and B. Both haplotypes A and B contain the framework
genes 3DL3, 3DP1, 2DL4, and 3DL2. Then there are the KIR genes
2DP1, 2DL1 and 2DS4 that are common for both haplotypes, but only
the haplotype A contains 2DL3, 3DL1 and 2DS4. Haplotype B is more
variable and can contain the KIR genes 2DS1, 2DS2, 2DS3, 2DS4,
2DS5, 3DS1, 2DL2 and 2DL5 (apart form the aforementioned framework
genes). In more than 96% of the worldwide global population the A
haplotype at KIR gene cluster contains the KIR genes 3DL3, 2DL3,
2DP1, 2DL1, 3DP1, 2DL4, 3DL1, 2DS4 and 3DL2 (see also:
www.allelfrequencies.net).
[0202] The novel KIR haplotype model assumes that haplotype A and B
are present on the two different chromosomes. Therefore any
individual can represent an AA, AB or BB genotype. Based on the
genes that are present in the DNA sample of that individual, one
can conclude which haplotypes are present and the positive genes
from the assay can be divided over both haplotypes according to the
rules that certain KIR genes are present only in one of the
haplotypes A or B, essentially as was mentioned above.
[0203] For the SSP PCR data the two haplotype models are shown to
interpret possible CNV results, resp. the conventional KIR
haplotype model in FIG. 11B1 and 12B1 and the novel KIR haplotype
model in FIG. 11B2 and 12B2. FIG. 11B3 and 12B3 show the results of
our MLPA data with the extended probe sets 1 & 2 compared with
both the SSP PCR data according to the conventional KIR haplotype
model and with the novel KIR haplotype model.
[0204] In conventional KIR haplotype model in FIGS. 11B1 and 12B1
the KIR gene region is described by framework genes (3DL3, 3DP1,
2DL4 and 3DL2), genes that can be present in both A and B
haplotypes (2DP1, 2DL1 and 2DS4) and haplotype-specific genes. The
KIR genes 2DL3, 3DL1 and 2DS4 are specific for haplotype A. while
the KIR genes 2DL5, 2DS1, 2DS2, 2DS3, 2DS5, 3DS1 and 2DL2 are
specific for haplotype B. The haplotype A is constant to a high
degree. In more than 96% of the global population haplotype A
consists of 3DL3, 2DL3, 2DP1, 2DL1, 3DP1, 2DL4, 3DL1, 2DS4 and 3DL2
(www.allelefrequencies.net). Haplotype B is more variable and
carries more activating KIR genes.
[0205] FIGS. 11B2 and 12B2 show the interpretation for the
respective families based on the novel KIR haplotype model and
SSP-PCR data from CEPH-IHWG.
[0206] FIGS. 11B3 and 12B3 show the copy number variation for the
respective families. In table 3 Copy number variation of KIR genes
by MLPA is determined by 2 probes for each gene, except for the
N-variant genes (single probe detection by definition), including
those genes marked by an asterisk.
[0207] For the 3DP1v gene variant a combination of 3 probes has
been designed. CNV can be deduced from a comparison between the
results for the probes for 2DL5, 2DL5a and 2DL5b.
[0208] The 2DS4N KIR probe is designed to detect the KIR-2DS4
deletion-variant genes *003 to *009, while SSP-PCR only detects
2DS4 variant *003 (designated 1D).
[0209] In FIG. 12B3 KIR3DP1 variants are detected using MLPA (table
3), whereas KIR3DP1 variants are not detected when SSP-PCR is used.
SSP-PCR of KIR3DP1v results in a band of 1672 bp that is obtained
from the 3DP1 gene. Because this is a large fragment which are
known to be difficult to detect. Therefore, a DNA sample can be
positive for KIR3DP1v when MLPA is used but appear to be negative
for KIR3DP1v when SSP-PCR is used.
CONCLUSION
[0210] Extended probe set 1 contains the probes 2DL1-5, 2DS1-5,
3DL1-3, 3DS1, 2DP1 and 3DP1, in total 20 probes. Extended probe set
2 contains the probes 2DL1-5, 2DS1-5, 3DL1-3, 3DS1, 2DP1 and 3DP1,
in total 20 probes. Together these two probe sets are accurate for
the typing of all 17 KIR genes, and 7 variant KIR gene variants
(i.e. 2DL5a, 2DL5b, 3DP1v, and the null-variants 2DL4N, 3DL1N,
3DS1N, and 2DS4N), and for quantifying relative copy numbers of at
least all 17 different KIR genes, and 4 null-variant (2DL4N, 3DL1N,
3DS1N, and 2DS4N) (see FIG. 13).
Example 3
[0211] The advantage of probe sets comprising three probe parts
according to the present invention is that at least two different
SNPs can be detected with one probe set. For instance, in a
probeset consisting of three probe parts two sites for ligation are
preferably present. A left probe part and middle probe part are
ligated and additionally a middle probe part and right probe part
are ligated. At each ligation site a SNP can be detected. With
conventional MLPA probe sets, consisting of two half probes, only
one SNP can be detected per probe set, because only one site for
ligation is present.
[0212] In this Example detection of the Null allele of KIR3DL1 with
a probeset consisting of three probes (one left probe part, one
middle probe part and one right probe part) is described. This
example is illustrated in FIG. 1C.
Materials & Methods
[0213] The null allele, called KIR3DL1*024N, is discriminated from
KIR3DL1 using three probes of the invention. Partial probes (probe
numbers as depicted in FIG. 3C) used in this example are:
TABLE-US-00001 711A - KIR3DL1 WT Left probe part: 5'-PO4
GGTTCCCTAAGGGTTGGACCCCTCACGCCTCGTTGGACA-3' 711D - KIR3DL1*024N Left
probe part: 5'-PO4-GGGTTCCCTAAGGGTTGGACAAGGACCCCTCACGCCTCGTTGG
AC-3' 711B - KIR3DL1 Middle probe part:
5'-PO4-GATCCATGATGGGGTCTCCAAGGCCAATTTCTCCATCGGTCCC ATGATGCT-3' 711C
- KIR3DL1 Right probe part:
5'-PO4-GCCCTTGCAGGGACCTACAGATGCTACGGTTCTGGTCTAGATT
GGATCTTGCTGGCAC-3'
[0214] For DNA selection/isolation, probe design, MLPA reaction,
electrophoresis and analysis see materials & methods of example
1.
[0215] With these partial probes 2 probe sets can be formed. Those
two probe sets consist of different left probe parts, but share the
middle and right probe parts.
Results and Discussion
[0216] The final base of middle probe part 711B is a thymine. This
thymine is specific for KIR3DL1 genes while all other KIR genes
have a different base at this position. Therefore, with probe part
711B KIR3DL1 is discriminated from other KIR genes. Ligation
between the middle probe part (711B) and right probe part (711C)
will only occur when KIR3DL1 genes are present. The final base of
left probe part 711A is an adenine. This base is present in
wildtype KIR3DL1 gene but deleted in the KIR3DL1 null allele,
KIR3DL1*024N. Thus, probe part 711A containing an adenine at the
final base position is specific for the wildtype KIR3DL1 gene and
ligation between the 711A left probe part and the middle probe part
(711B) will only occur if the KIR3DL1 wildtype gene is present. In
left probe part 711D the final adenine is removed. Thus, probe part
711D is specific for null allele KIR3DL1*024N and ligation between
the 711D left probe part and the middle probe part (711B) will only
occur if KIR3DL1*024N is present.
[0217] Thus these two probe sets each detect 2 SNPs, namely those
SNPs that are specific for KIR3DL1 wildtype gene and null allele
KIR3DL1*024N because both the left probe part and the middle probe
part are SNP-specific.
TABLE-US-00002 TABLE 1 KIR genes and proteins names, adapted from
KIR Nomenclature report 2002 (Marsh et al, 2002). Gene Protein
symbol symbol Aliases KIR2DL1 KIR2DL1 cl-42, nkat1, 47.11, p58.1,
CD158a KIR2DL2 KIR2DL2 cl-43, nkat6, CD158b1 KIR2DL3 KIR2DL3 c1-6,
nkat2, nkat2a, nkat2b, p58, CD158b2 KIR2DL4 KIR2DL4 103AS, 15.212,
CD158d KIR2DL5A KIR2DL5A KIR2DL5.1, CD158f KIR2DL5B KIR2DL5B
KIR2DL5.2, KIR2DL5.3, KIR2DL5.4 KIR2DS1 KIR2DS1 EB6ActI, EB6ActII,
CD158h KIR2DS2 KIR2DS2 cl-49, nkat5, 183ActI, CD158j KIR2DS3
KIR2DS3 nkat7 KIR2DS4 KIR2DS4 cl-39, KKA3, nkat8, CD158i KIR2DS5
KIR2DS5 nkat9, CD158g KIR2DP1 KIR2DP1 KIRZ, KIRY, KIR15, KIR2DL6
KIR3DL1 KIR3DL1 cl-2, NKB1, cl-11, nkat3, NKB1B, AMB11, KIR,
CD158e1 KIR3DL2 KIR3DL2 cl-5, nkat4, nkat4a, nkat4b, CD158k KIR3DL3
KIR3DL3 KIRC1, KIR3DL7, KIR44, CD158z KIR3DS1 KIR3DS1 nkat10,
CD158e2 KIR3DP1 KIR3DP1 KIRX, KIR48, KIR2DS6, KIR3DS2P, CD158c
TABLE-US-00003 TABLE 2 Number of currently known alleles for each
KIR gene and the different protein products they encode (IPD KIR
database, http://www.ebi.ac.uk/ipd/kir). Gene 2DL1 2DL2 2DL3 2DL4
2DL5 2DS1 2DS2 2DS3 Alleles 25 11 9 25 21 12 12 9 Proteins 28 7 8
12 11 8 6 3 Gene 2DS4 2DS5 3DL1 3DS1 3DL2 3DL3 2DP1 3DP1 Alleles 20
12 52 14 45 55 5 8 Proteins 13 9 46 12 40 31 0 0
TABLE-US-00004 TABLE 3 KIRs and their cognate ligands (Carrington
et al, 2003; Middleton et al, 2005; Du et al, 2007). The ligands of
the other KIRs are unknown or uncertain. Inhibitory KIRs Ligands
Activating KIRs Ligands 2DL1 HLA-C group 2, 2DS1 HLA-C group 2
allotypes allotypes Cw1, 4, 5, 6, 17, 18 Cw1, 4, 5, 6, 17, 18 2DL2
and 2DL3 HLA-C group 1, 2DS2 HLA group 1, allotypes allotypes Cw1,
3, 7, 8, 13, 14 Cw1, 3, 7, 8, 13, 14 2DL4 HLA-G 2DS4 HLA-C 3DL1
HLA-B, Bw4 3DS1 HLA-B, Bw4 3DL2 HLA-A3 and A11 allotypes
TABLE-US-00005 TABLE 4 The KIR Reference Panel I from the IHWG
(http://www.ihwg.org/cellbank/dna/refpan_nkkir_table.html). 2DS4
indicates all alleles except KIR2DS4*003 and 1D indicates only
KIR2DS4*003. 3DP1 indicates KIR3DP1*003 (deletion of exon 2) only
and 3DP1v indicates all alleles except KIR3DP1*003 ##STR00001##
##STR00002## ##STR00003## Note: "1" = presence of KIR gene "0" =
absence of KIR gene shaded cells (N = 16) represent four
informative families selected for the Phase II reference panel
TABLE-US-00006 TABLE 5 The 17 KIR probes that have been designed
and tested for probe set 1. The size of the complete MLPA probe and
the size of the separate probe parts and the concentration used are
listed in this table. Size Probe Size Concentration Code Probe [bp]
Part [bp] (fmol) 420A 2DL2 96 Left 48 1 420B Right 48 512A 3DL3 100
Left 50 1 512B Right 50 540A 2DS3 108 Left 54 10 540B Right 54 404A
3DL2 112 Left 56 1 404B Right 56 405A 2DP1 121 Left 65 1 405B Right
56 406A 3DP1 125 Left 66 1 406B Right 59 504A 2DS4 137 Left 61 1
504B Right 76 408A 2DL5 142 Left 57 1 408B Middle 32 408C Right 53
514A 3DL1 149 Left 74 1 514B Right 75 526A 2DS2 154 Left 57 1 526B
Middle 34 526C Right 63 507A 2DL5A 165 Left 66 1 507B Middle 32
507C Right 67 419A 2DL4 170 Left 59 1 419B Middle 54 419C Right 57
528A 2DS5 185 Left 67 1 528B Middle 47 528C Right 71 413A 2DL1 189
Left 72 1 413B Middle 64 413C Right 53 416A 2DS1 195 Left 78 10
416B Middle 67 416C Right 50 415A 2DL3 213 Left 75 10 415B Middle
69 415C Right 69 418A 3DS1 218 Left 81 10 418B Middle 64 418C Right
73
TABLE-US-00007 TABLE 6 The 17 KIR probes that have been designed
and tested for probe set 2. The size of the complete MLPA probe and
the size of the separate probe parts and the concentration used are
listed in this table. Size Probe Size Concentration Code Probe [bp]
Part [bp] (fmol) 543A 2DS1 96 Left 48 10 543B Right 48 544A 2DS2
100 Left 50 1 544B Right 50 537A 2DL5 108 Left 54 1 537B Right 54
513D 2DS3 112 Left 52 10 513B Right 60 518A 3DP1 121 Left 61 1 518B
Right 60 542A 2DP1 125 Left 60 1 542B Right 65 541A 3DS1 134 Left
67 10 541B Right 67 524A 2DS4 137 Left 66 10 524B Right 71 545A
2DS5 144 Left 68 10 545B Right 76 409A 3DL1 149 Left 60 10 409B
Middle 34 409C Right 55 506A 3DL3 154 Left 54 10 506B Middle 48
506C Right 52 507A 2DL5A 165 Left 66 1 507B Middle 32 507C Right 67
539A 2DL2 170 Left 60 1 539B Middle 46 539C Right 64 525A 2DL1 190
Left 64 10 525B Middle 62 525C Right 64 538A 3DL2 r 195 Left 70 1
538B Middle 60 538C Right 65 417A 2DL3 213 Left 75 10 417B Middle
69 417C Right 69 517A 2DL4 218 Left 73 10 517B Middle 68 517C Right
77
TABLE-US-00008 TABLE 7 The control probes used in the two probes
sets. The size of the complete MLPA probe and the size of the
separate probe parts and the concentration used for the probe sets
are listed in this table. Size Probe Size Concentration Code Probe
(Gene) [bp] part [bp] (fmol) 201 Ctrl 2 (FGF3) 92 Left 45 1 Right
47 202 Ctrl 3 (BCAS4) 104 Left 52 1 Right 52 203 Ctrl 4 (LMNA) 116
Left 58 1 Right 58 204 Ctrl 5 (PARK2) 130 Left 44 3 Middle 41 Right
45 205 Ctrl 7 (MSH6) 160 Left 59 1 Middle 42 Right 59 206 Ctrl 8
(GALT) 175 Left 58 1 Middle 59 Right 58 207 Ctrl 9 (SPG4) 180 Left
60 1 Middle 60 Right 60 210 Ctrl 1 (IL-4) 208 Left 73 1 Middle 69
Right 66 209 Ctrl 10 (NF2) 223 Left 78 1 Middle 69 Right 76
TABLE-US-00009 TABLE 8 The competitors of the control probes. The
size of the competitor, the part of the control probes used and
concentration used for the probe sets are listed in this table.
code gene length [bp] probe part Concentration (fmol) 201X Ctrl 2
(FGF3) 30 Left 10 202X Ctrl 3 (BCAS4) 30 Left 10 203X Ctrl 4 (LMNA)
30 Left 3 205X Ctrl 7 (MSH6) 50 Left 0 207X Ctrl 9 (SPG4) 50 Left
1
TABLE-US-00010 TABLE 9 KIR gene frequencies in the Caucasian
population. The frequencies are derived from several studies
performed worldwide in the Caucasian population and are available
on www.allelfrequencies.net. KIR2DL1 KIR2DL2 KIR2DL3 KIR2DL4
KIR2DL5 KIR2DS1 KIR2DS2 KIR2DS3 88-100% 39-63% 57-94% 100% 36-61%
27-49% 25-63% 19-42% KIR2DS4 KIR2DS5 KIR3DL1 KIR3DL2 KIR3DL3
KIR3DS1 KIR2DP1 KIR3DP1 87-98% 21-46% 76-98% 99-100% 99-100% 26-50%
94-100% 97-100%
TABLE-US-00011 TABLE 10 KIR alleles detected by the probes and the
coverage of the total KIR alleles, except for 3DP1v, by probe sets
1 and 2, as depicted in FIG. 3A and 3B. All KIR alleles including
3DP1v are also detected by extended probe sets 1 and 2, as depicted
in FIG. 3C and 3D Coverage lower then 100% are caused by gene
variants that are present in the target sequence to which the
probes bind. The alleles shown here that can be detected by the
probes are generated with the primer or probe blast tool on the IPD
KIR database. The percentage of the total KIR alleles that can be
covered by the probes is calculated by dividing the number of
alleles for each probe by the number of total alleles that is
reported on the website. Certain alleles are underlined where the
coverage of both probe sets is not 100% due to gene variants
present in the target sequence. Probe set Probe set 1 Probe set 2 1
+ 2 PROBE ALLELES COVERAGE PROBE ALLELES COVERAGE COVERAGE 2DL1
2DL1*001 2DL1*00402 100% 2DL1 2DL1*001 2DL1*00402 100% 100%
2DL1*002 2DL1*005 2DL1*002 2DL1*005 2DL1*00301 2DL1*006 2DL1*00301
2DL1*006 2DL1*0030201 2DL1*007 2DL1*0030201 2DL1*007 2DL1*0030202
2DL1*008 2DL1*0030202 2DL1*008 2DL1*00303 2DL1*009 2DL1*00303
2DL1*009 2DL1*0040101 2DL1*010 2DL1*0040101 2DL1*010 2DL1*0040102
2DL1*0040102 2DL2 2DL2*001 2DL2*004 100% 2DL2 2DL2*001 2DL2*003 80%
100% 2DL2*002 2DL2*005 2DL2*002 2DL2*005 2DL2*003 2DL3 2DL3*001
2DL3*004 86% 2DL3 2DL3*001 2DL3*005 100% 100% 2DL3*002 2DL3*005
2DL3*002 2DL3*006 2DL3*003 2DL3*006 2DL3*003 2DL3*007 2DL3*004 2DL4
2DL4*00101 2DL4*00501 54% 2DL4 2DL4*00101 2DL4*00601 100% 100%
2DL4*00102 2DL4*00601 2DL4*00102 2DL4*00602 2DL4*00105 2DL4*00602
2DL4*0010301 2DL4*007 2DL4*00201 2DL4*007 2DL4*0010302 2DL4*0080101
2DL4*00202 2DL4*0080101 2DL4*00104 2DL4*0080102 2DL4*003
2DL4*0080201 2DL4*00105 2DL4*0080103 2DL4*004 2DL4*011 2DL4*00201
2DL4*0080104 2DL4*00202 2DL4*0080201 2DL4*00203 2DL4*0080202
2DL4*003 2DL4*009 2DL4*004 2DL4*010 2DL4*00501 2DL4*011 2DL4*00502
2DL4*012 2DL5 2DL5A*0010101 2DL5B*003 100% 2DL5 2DL5A*0010101
2DL5B*00601 54% 100% 2DL5A*0010102 2DL5B*004 2DL5A*0010102
2DL5B*007 2DL5A*0050101 2DL5B*00601 2DL5B*003 2DL5B*00801
2DL5A*0050102 2DL5B*007 2DL5B*004 2DL5B*0020101 2DL5B*00801
2DL5B*0020102 2DL5B*009 2DL5B*0020103 2DL5A 2DL5A*0010101
2DL5A*0050101 100% 2DL5A Same probe as in probe set 1. 100% 100%
2DL5A*0010102 2DL5A*0050102 3DP1*004 14% 3DP1v 2DS1 No match found
in the KIR 2DS1 No match found in the KIR database. BLAST result in
match database. Probe designed on with KIR2DS1v alias KIR2DS1*002
intron 6. 2DS2 2DS2*0010101 2DS2*002 90% 2DS2 No match found in the
KIR 90% 2DS2*0010102 2DS2*003 database. Probe designed on
2DS2*0010103 2DS2*004 intron 2 and 3. 2DS2*00102 2DS2*005
2DS2*00103 2DS3 2DS3*00101 2DS3*002 100% 2DS3 2DS3*00101 2DS3*002
100% 100% 2DS3*00102 2DS3*003N 2DS3*00102 2DS3*003N 2DS3*00103
2DS3*004 2DS3*00103 2DS3*004 2DS3*00104 2DS3*00104 2DS4
2DS4*0010101 2DS4*003 100% 2DS4 2DS4*0010101 2DS4*003 80% 100%
2DS4*0010102 2DS4*004 2DS4*0010102 2DS4*006 2DS4*0010103 2DS4*006
2DS4*0010103 2DS4*007 2DS4*00102 2DS4*007 2DS4*00102 2DS4*009
2DS4*00103 2DS4*009 2DS5 2DS5*001 2DS5*004 100% 2DS5 2DS5*001
2DS5*004 100% 100% 2DS5*0020101 2DS5*005 2DS5*0020101 2DS5*005
2DS5*0020102 2DS5*006 2DS5*0020102 2DS5*006 2DS5*0020103 2DS5*007
2DS5*0020103 2DS5*007 2DS5*003 2DS5*008 2DS5*003 2DS5*008 3DL1
3DL1*00101 3DL1*027 78% 3DL1 3DL1*00101 3DL1*021 41% 88% 3DL1*00102
3DL1*028 3DL1*002 3DL1*022 3DL1*002 3DL1*029 3DL1*00401 3DL1*023
3DL1*00401 3DL1*030 3DL1*00402 3DL1*024N 3DL1*00402 3DL1*031
3DL1*00501 3DL1*025 3DL1*00501 3DL1*032 3DL1*00502 3DL1*026
3DL1*00502 3DL1*033 3DL1*006 3DL1*027 3DL1*007 3DL1*034 3DL1*007
3DL1*028 3DL1*008 3DL1*035 3DL1*008 3DL1*029 3DL1*009 3DL1*036
3DL1*009 3DL1*030 3DL1*01501 3DL1*037 3DL1*01502 3DL1*038 3DL1*016
3DL1*039 3DL1*01701 3DL1*040 3DL1*01702 3DL1*041 3DL1*018 3DL1*042
3DL1*024N 3DL1*043 3DL1*025 3DL1*044 3DL1*026 3DL1*057 3DL2
3DL2*00101 3DL2*00902 47% 3DL2 3DL2*00101 3DL2*010 45% 61% 3DL2*002
3DL2*013 3DL2*002 3DL2*011 3DL2*00301 3DL2*014 3DL2*00301 3DL2*012
3DL2*004 3DL2*016 3DL2*004 3DL2*013 3DL2*005 3DL2*017 3DL2*005
3DL2*015 3DL2*0070101 3DL2*018 3DL2*006 3DL2*016 3DL2*0070102
3DL2*019 3DL2*0070101 3DL2*020 3DL2*008 3DL2*020 3DL2*0070102
3DL2*021 3DL2*00901 3DL2*021 3DL2*008 3DL3 3DL3*00101 3DL3*01102
75% 3DL3 3DL3*00101 3DL3*01303 100% 100% 3DL3*00102 3DL3*012
3DL3*00102 3DL3*01304 3DL3*00103 3DL3*01301 3DL3*00103 3DL3*01305
3DL3*00201 3DL3*01303 3DL3*00201 3DL3*01306 3DL3*00203 3DL3*01304
3DL3*00202 3DL3*01307 3DL3*00204 3DL3*01401 3DL3*00203 3DL3*01401
3DL3*00205 3DL3*01403 3DL3*00204 3DL3*01402 3DL3*00207 3DL3*01405
3DL3*00205 3DL3*01403 3DL3*0030101 3DL3*015 3DL3*00206 3DL3*01404
3DL3*0030102 3DL3*016 3DL3*00207 3DL3*01405 3DL3*00401 3DL3*017
3DL3*0030101 3DL3*015 3DL3*00402 3DL3*018 3DL3*0030102 3DL3*016
3DL3*005 3DL3*020 3DL3*00401 3DL3*017 3DL3*00601 3DL3*021
3DL3*00402 3DL3*018 3DL3*00602 3DL3*022 3DL3*005 3DL3*019
3DL3*00801 3DL3*023 3DL3*00601 3DL3*020 3DL3*00802 3DL3*024
3DL3*00602 3DL3*021 3DL3*00901 3DL3*025 3DL3*007 3DL3*022
3DL3*00902 3DL3*026 3DL3*00801 3DL3*023 3DL3*010 3DL3*028
3DL3*00802 3DL3*024 3DL3*01101 3DL3*00901 3DL3*025 3DL3*00902
3DL3*026 3DL3*010 3DL3*027 3DL3*01101 3DL3*028 3DL3*01102 3DL3*029
3DL3*012 3DL3*030 3DL3*01301 3DL3*031 3DL3*01302 3DS1 3DS1*010
3DS1*046 71% 3DS1 3DS1*010 3DS1*045 71% 86% 3DS1*01301 3DS1*047
3DS1*011 3DS1*046 3DS1*01302 3DS1*048 3DS1*012 3DS1*047 3DS1*014
3DS1*049N 3DS1*01301 3DS1*048 3DS1*045 3DS1*055 3DS1*01302
3DS1*049N 2DP1 2DP1*00101 2DP1*0020102 100% 2DP1 2DP1*00101
2DP1*0020102 100% 100% 2DP1*00102 2DP1*003 2DP1*00102 2DP1*003
2DP1*0020101 2DP1*0020101 3DP1 3DP1*001 3DP1*004 100% 3DP1 No match
found on the KIR 100% 3DP1*002 3DP1*005 database. 3DP1*00301
3DP1*006 Detects deletion of exon 2. 3DP1*00302
TABLE-US-00012 TABLE 11 Verification of KIR MLPA probe set 1 on 11
cell lines KIR-genotyped by the 10.sup.th IHW. KIR genotyped Cell
lines by the 10.sup.th IHW, results of probes set1. CODE NAME 2DL1
2DL2 2DL3 2DL4 2DL5 2DL5A 2DS1 2DS2 2DS3 2DS4 2DS5 3DL1 3DL2 3DL3
3DS1 2DP1 3DP1 231 JVM 1 1 1 1 0 4 0 1 0 1 0 1 1 4 0 4 4 240 T7507
1 1 1 1 1 4 1 1 1 1 0 1 1 4 1 4 4 343 OLGA 1 0 1 1 1 4 1 2 0 1 1 1
1 4 1 4 4 423 SAVC 1 0 1 1 0 4 0 2 0 1 0 1 1 4 0 4 4 712 JBUSH 1 0
1 1 0 4 0 2 0 1 0 1 1 4 0 4 4 723 BM16 1 0 1 1 0 4 0 2 0 1 0 1 1 4
0 4 4 773 LBUF ##STR00004## 1 2 1 3 4 3 1 3 1 3 1 1 4 3 4 4 931
AMALA 1 1 1 1 1 4 1 1 0 1 1 1 1 4 1 4 4 1042 BM90 1 1 1 1 1 4 1 1 1
1 1 1 1 4 1 4 4 1102 TAB089 1 0 1 1 0 4 0 2 0 1 0 1 1 4 0 4 4 122
KAS116 1 0 1 1 0 4 0 2 0 1 0 1 1 4 0 4 4 0 = negative by MLPA and
10th IHW 1 = positive by MPLA and 10th IHW 2 = positive by MLPA and
negative by 10th IHW 3 = negative by MLPA and positive by 10th IHW
4 = not typed by 10th IHW but positive by MLPA ##STR00005##
negative by two laboratories and positive typed by one
TABLE-US-00013 TABLE 12 Verification of KIR MLPA probe set 1 on 5
PCR-SSP KIR typed samples. PCR-SSP KIR typed DNA, results of probe
set 1. sample 2DL1 2DL2 2DL3 2DL4 2DL5 2DL5A 2DS1 2DS2 2DS3 2DS4
2DS5 3DL1 3DL2 3DL3 3DS1 2DP1 3DP1 33_7536 1 0 1 1 1 3 1 2 1 1 1 1
1 1 1 1 1 33_8025 1 0 1 1 0 3 0 2 0 1 0 1 1 1 0 1 1 33_8037 1 0 1 1
1 3 1 2 0 1 1 1 1 1 1 1 1 33_8588 0 1 0 1 0 3 0 1 0 1 0 1 1 1 0 0 1
33_9097 1 1 0 1 1 3 1 1 1 0 1 0 1 1 1 1 1 0 = negative by MLPA and
SSP 1 = positive by MPLA and SSP 2 = positvie by MLPA and negative
by SSP 3 = positive by MLPA not typed by SSP
TABLE-US-00014 TABLE 13 Verification of KIR MLPA probe set 2 on 11
cell lines KIR-genotyped by the 10.sup.th IHW. KIR genotyped Cell
lines by the 10.sup.th IHW, results of probe set2. CODE NAME 2DL1
2DL2 2DL3 2DL4 2DL5 2DL5A 2DS1 2DS2 2DS3 2DS4 2DS5 3DL1 3DL2 3DL3
3DS1 2DP1 3DP1 231 JVM 1 1 1 1 0 4 0 1 0 1 2 1 1 4 2 4 4 240 T7507
1 1 1 1 1 4 1 1 1 1 2 1 1 4 1 4 4 343 OLGA 1 0 1 1 1 4 1 0 0 1 2 1
1 4 1 4 4 423 SAVC 1 0 1 1 0 4 0 0 0 1 2 1 1 4 2 4 4 712 JBUSH 1 0
1 1 0 4 0 0 0 1 2 1 1 4 2 4 4 723 BM16 1 0 1 1 0 4 2 0 0 1 2 1 1 4
2 4 4 773 LBUF ##STR00006## 1 2 1 3 4 1 1 3 1 1 1 1 4 1 4 4 931
AMALA 1 1 1 1 1 4 1 1 0 1 1 1 1 4 1 4 4 1042 BM90 1 1 1 1 1 4 1 1 1
1 1 1 1 4 1 4 4 1102 TAB089 1 0 1 1 0 4 2 0 0 1 2 1 1 4 2 4 4 122
KAS116 1 0 1 1 0 4 2 0 0 1 2 1 1 4 2 4 4 0 = negative by MLPA and
10th IHW 1 = positive by MPLA and 10th IHW 2 = positive by MLPA and
negative by 10th IHW 3 = negative by MLPA and positive by 10th IHW
4 = not typed by 10th IHW but positive by MLPA ##STR00007##
negative by two laboratories and positive typed by one
TABLE-US-00015 TABLE 14 Verification of KIR MLPA probe set 21 on 5
PCR-SSP KIR typed samples. PCR-SSP KIR typed patients, results of
probe set 2. sample 2DL1 2DL2 2DL3 2DL4 2DL5 2DL5A 2DS1 2DS2 2DS3
2DS4 2DS5 3DL1 3DL2 3DL3 3DS1 2DP1 3DP1 33_7536 1 0 1 1 1 4 1 0 1 1
1 1 1 1 1 1 1 33_8025 1 0 1 1 0 4 0 0 0 1 2 1 1 1 2 1 1 33_8037 1 0
1 1 1 4 1 0 0 1 1 1 1 1 1 1 1 33_8588 2 1 0 1 0 4 0 1 0 1 2 1 1 1 2
0 3 33_9097 1 1 0 1 1 4 1 1 1 0 1 2 1 1 1 1 1 0 = negative by MLPA
and SSP 1 = positive by MPLA and SSP 2 = positive by MLPA and
negative by SSP 3 = negative by MLPA and positive by SSP 4 =
positive by MLPA not typed by SSP
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Sequence CWU 1
1
163119DNAArtificialprimer 1gggttcccta agggttgga
19223DNAArtificialprimer 2tctagattgg atcttgctgg cac
23318DNAArtificialprimer 3gggttcccta agggttgg
18439DNAArtificialKIR3DL1 WT Left probe part 4ggttccctaa gggttggacc
cctcacgcct cgttggaca 39545DNAArtificialKIR3DL1*024N Left probe part
5gggttcccta agggttggac aaggacccct cacgcctcgt tggac
45651DNAArtificialKIR3DL1 Middle probe part 6gatccatgat ggggtctcca
aggccaattt ctccatcggt cccatgatgc t 51758DNAArtificialKIR3DL1 Right
probe part 7gcccttgcag ggacctacag atgctacggt tctggtctag attggatctt
gctggcac 58824DNAArtificialprobe KIR2DS5 8cgcatgacac aagacctggc
aggg 24924DNAArtificialprobe KIR2DS3 9cgcatgaggc aagacctggc aggg
241024DNAArtificialprobe KIR2DP1 10cccatgatgg aagacctggc aggg
241124DNAArtificialprobe KIR2DL1 11cgcatgacgc aagacctggc aggg
241224DNAArtificialprobe KIR2DS1 12cgcatgaagc aagacctggc aggg
241324DNAArtificialprobe KIR2DS4 13cccatgatgc ctgtccttgc agga
241424DNAArtificialprobe KIR3DS1 14tccatgatgc gtgcccttgc aggg
241524DNAArtificialprobe KIR2DL3 15cccatgatgc aagaccttgc aggg
241624DNAArtificialprobe KIR3DP1 16cgcatgacgc aagaccttgc aggg
241724DNAArtificialprobe KIR3DL2 17cccttgatgc ctgtccttgc agga
241824DNAArtificialprobe KIR3DL3 18cccatgacac ctgcccttgc aggg
241948DNAArtificialprobe 19gggttcccta agggttggac tgaccttggg
ccctgcagag aacctaca 482048DNAArtificialprobe 20ttcatgggcc
tccccctccc tggatgtcta gattgatctt gctggcac 482150DNAArtificialprobe
21gggttcccta agggttggat agatgcttcg gctctttccg tgccctgccc
502250DNAArtificialprobe 22cacgcgtggt cagacccgag tgacccgtct
agattggatc ttgctggcac 502354DNAArtificialprobe 23gggttcccta
agggttggac catcacgatg tccagagggt cactgggagc tgaa
542454DNAArtificialprobe 24aactgatagg gggagtgagg aacagagacc
gtctagattg gatcttgctg gcac 542555DNAArtificialprobe 25gggttcccta
agggttggaa tccaccctaa ggtttgggga aggactcacc catga
552656DNAArtificialprobe 26gtggccaggc cccctgcagc aagaagaacc
ctgtctagat tggatcttgc tggcgc 562765DNAArtificialprobe 27gggttcccta
agggttggac ccaaggtggt caggacaagc ccttgctgtc tgcctggccc 60agctc
652856DNAArtificialprobe 28tgtggtgcct ccaggacatg tgattcttcg
gtgtctagat tggatcttgc tggcgc 562966DNAArtificialprobe 29gggttcccta
agggttggac accatgatca ccagggggtt gctgggtgct gaccacccag 60tgagga
663059DNAArtificialprobe 30agtgtgggtg tgaaccccga catctgtagg
tccctgtcta gattggatct tgctggcgc 593161DNAArtificialprobe
31gggttcccta agggttggac tcccctctct gtgcagaagg aagtgctcaa acatgacatc
60c 613276DNAArtificialprobe 32gaccaacatt gcaggatgac tgtctcttct
gatttcacca ggtgacctgg gagtctagat 60tggatcttgc tggcac
763357DNAArtificialprobe 33gggttcccta agggttggac tcaggtgtga
ggggagctgt gacaaggaag aacctcc 573432DNAArtificialprobe 34ctgaggaaac
tgcctcttct tccaggtcta tt 323553DNAArtificialprobe 35tgggaaacct
tcactctcag cccagccggg tctagattgg atcttgctgg cgc
533674DNAArtificialprobe 36gggttcccta agggttggac gtgttcttat
ctaggatact ccaaggccaa tttctccatc 60ggtcccatga tgct
743775DNAArtificialprobe 37tgcccttgca gggacctaca gatgctacgg
ttctgttctc tcgtcagacg tgtctagatt 60ggatcttgct ggcac
753869DNAArtificialprobe 38gggttcccta agggttggac ggggcgcggc
tgcctgtctg caccggcagc accatgtcgc 60tcatggtca
693932DNAArtificialprobe 39tcagcatggc gtgtgttggt gagtcctgga aa
324067DNAArtificialprobe 40tgaccatgag cgacatggtg ctgccggtgc
agacgggagg ttggtctaga ttggatcttg 60ctggcac 674159DNAArtificialprobe
41gggttcccta agggttggac ctgcttcaga acatggctct ctgctgggga gacacccaa
594254DNAArtificialprobe 42tctgcaggcc catagtgtaa ccctggtgct
ccttcccttc caggactcac caag 544357DNAArtificialprobe 43acatgccagg
atgatgaccg tgggtgacat ggagtctaga ttggatcttg ctggcac
574467DNAArtificialprobe 44gggttcccta agggttggac cgagtaaacc
ggaaaatttt catctgcaca gagaggggac 60gtttaac 674547DNAArtificialprobe
45cacactttgc gcctcattgg agagcacatt gatggggtct ccaaggg
474671DNAArtificialprobe 46caacttctcc atcggtcgca tgacacaaga
cctggcatag cgaatacgtc tagattggat 60cttgctggca c
714772DNAArtificialprobe 47gggttcccta agggttggac taccccatcg
ctcttcatgc tggatcattc actctgcatc 60ccaatgacaa tg
724864DNAArtificialprobe 48agaagaaagt ctggacactc tcacctatga
tcacgatgtc cagagggtca ctgggagctg 60acac 644953DNAArtificialprobe
49ctgatagggg gagtgagtaa cagaaccgta gtctagattg atcttgctgg cac
535078DNAArtificialprobe 50gggttcccta agggttggac acagggccca
tgaaaaggct gttccagaat attatgttgt 60agagctcagg gacaggca
785167DNAArtificialprobe 51ccccatcttc cttttacaga ctgaagttgt
taaacccaag ataagaatga cactgaagaa 60tcacata 675250DNAArtificialprobe
52tcctggaggc accacagggc ttggccagtc tagattgatc ttgctggcgc
505370DNAArtificialprobe 53gggttcccta agggttggac tgcacagttg
gatcactgcg ttttcacaca gagaaaaatc 60actcgccctt
705466DNAArtificialprobe 54ctcagaggcc caagacaccc ccaacagata
tcatcgtgta cacggaactt ccaaatgctg 60agccct 665567DNAArtificialprobe
55gatccaaagt tgtctcctgc ccatgagcac cacagtcagg ccttgtctag attgatcttg
60ctggcgc 675681DNAArtificialprobe 56gggttcccta agggttggac
ttgttcatca gaatcctgga gagagggaaa tgctgagtga 60gggagggtgc tcacattttt
c 815764DNAArtificialprobe 57aggactcttt gggaataaca ctagccacga
ggctgggccg aggagcacct acctcgctgt 60tcac 645874DNAArtificialprobe
58ttctgttccc tgcaggctct tggtccatta cagcagcatc tgtaggaaga cgtctagatt
60gatcttgctg gcgc 745950DNAArtificialprobe 59caygcgtggt cagacccgag
tgacccgtct agattggatc ttgctggcac 506070DNAArtificialprobe
60gggttcccta agggttggac yrcacagttg ratcactgcg ttttcacaca gagaaaaatc
60actcrccctt 706158DNAArtificialprobe 61gggttcccta agggttggac
ctgcagggga cgtgagggta cagttcagat tcaggcaa 586230DNAArtificialprobe
62cggtctgtga gctgaaggca ggggaaggga 306359DNAArtificialprobe
63atctggtgct ctctctagaa agtcctgcct ctgtggtcta gattggatct tgctggcac
596458DNAArtificialprobe 64gggttcccta agggttggac ctgcagggga
cgtgagggta cagttcagaa tcaggcaa 586544DNAArtificialprobe
65ttttggagca ccagcgatga aggagaaaga agggaaggat ggta
446657DNAArtificialprobe 66aagaggatga tggccactga gtacctaatc
acagtctaga ttggatcttg ctggcac 576740DNAArtificialprobe 67gggttcccta
agggttggac ccctcahgcc tcgytggaca 406851DNAArtificialprobe
68gatccatgat ggggtctcca aggccaattt ctccatyggt cccatgatgc t
516959DNAArtificialprobe 69tgcccttgca grgacctaca gatgctacgg
ttctggtcta gattggatct tgctggcac 597045DNAArtificialprobe
70gggttcccta agggttggac aaggacccct cacgcctcgt tggac
457180DNAArtificialprobe 71gggttcccta agggttggac atgttagcac
agattttagg catctcgtgt tcggataaaa 60atacatgaaa agtctttcac
807279DNAArtificialprobe 72gttagcacag attttaggca tcttgtgttc
gggaggttgg atctgagacg tgttgtgagt 60tggtcatagt gaaggacgt
797370DNAArtificialprobe 73gaggtgccaa ttctagtgag aacaatttcc
aggaagccgt gttccggtct agattggatc 60ttgctggcac
707453DNAArtificialprobe 74aactgatagg gggagtgagg aacagaaccg
tctagattgg atcttgctgg cac 537574DNAArtificialprobe 75ttctgttccc
tgcaggctct tggtccatta cagcagcatc tgtagaagac gtctagattg 60gatcttgctg
gcac 747654DNAArtificialprobe 76gggttcccta agggttggac aagggtgaga
ggcaggtctg tattctctca ccta 547748DNAArtificialprobe 77cgaccacgat
gtccagaggg tcactgggag ccgacaactc atagggta 487852DNAArtificialprobe
78agtgagtgac agaaccaaag catctgtagt ctagattgga tcttgctggc ac
527948DNAArtificialprobe 79cgaccacgat gtccagaggg tcactgggag
cygacaactc atagggta 488075DNAArtificialprobe 80gggttcccta
agggttggac tgctgccttg ggccagggac catcctgtct gtgaggaaca 60cacacctgag
tgctg 758169DNAArtificialprobe 81ccatcctgct tccccacatg gccctgagct
ctctggcctc tgcttcgtga gacttacttt 60ttttgttgc
698270DNAArtificialprobe 82agcaccagcg atgaaggaga aagaagagga
ggaggatgaa gaggatgtct agattggatc 60ttgctggcac
708350DNAArtificialprobe 83gggttcccta agggttggac cggccgagca
ccccagggtc ctctcttccc 508450DNAArtificialprobe 84agtttatgag
agactccctg acaggacgtc tagattgatc ttgctggcac
508574DNAArtificialprobe 85gggttcccta agggttggac atgtcctatg
atcctagagc cttagctgag gagcttcctg 60ctgatgatgg agat
748671DNAArtificialprobe 86aagcatggac agatgcagag agaagacgaa
gcttgggtgt gagggaggtc tagattggat 60cttgctggca c
718760DNAArtificialprobe 87gggttcccta agggttggac cagggaccta
cagatgctac ggttctgtta ctcactcccc 608865DNAArtificialprobe
88catcagttgt cagctcccag tgaccctctg gacatcgtca tgtctagatt ggatcttgct
60ggcac 658947DNAArtificialprobe 89gggttcccta agggttggac atcctgtgcg
ctgctgagct gagctcg 479050DNAArtificialprobe 90gtcgcggctg cctgtctgct
ccggcagtct agattggatc ttgctggcac 509161DNAArtificialprobe
91gggttcccta agggttggac gctgaggcct ggaaaggaat agagggaggg agtgccacat
60c 619260DNAArtificialprobe 92ctcctctcta aggtggcgcc tccttctccc
ccaggtgtct agattggatc ttgctggcac 609373DNAArtificialprobe
93gggttcccta agggttggac ccgcttagaa agaagaaatg gggagaatct tctgagcaca
60gggagggagg ggc 739468DNAArtificialprobe 94agctcaacat actcctctct
gaggcggcat ctccttctcc ccaaggtggt caggacaagc 60ccttctgc
689577DNAArtificialprobe 95tctgcctggc ccagcgctgt ggtgcctcaa
ggaggacacg tgactcttcg gtggtctaga 60ttggatcttg ctggcac
779660DNAArtificialprobe 96gggttcccta agggttggac tcagctcagg
tatgagggga gctatgacaa ggaagaacct 609734DNAArtificialprobe
97ccctgaggaa actgcctctt ctccttccag gtcc 349856DNAArtificialprobe
98atatgagaaa ccttctctct cagcccagcc gggtctagat tggatcttgc tggcac
569954DNAArtificialprobe 99gggttcccta agggttggac cgacctacac
atgcttcggc tctctccatg actc 5410054DNAArtificialprobe 100gggttcccta
agggttggac cgacctacac atgcttyrgc tctctccatg actc
5410154DNAArtificialprobe 101accctatgag tggtcagacc cgagtgaccc
gtctagattg gatcttgctg gcac 5410269DNAArtificialprobe 102gggttcccta
agggttggac ggggcgcggc tgcctgtctg caccggcagc accatgtcgc 60tcatggtca
6910332DNAArtificialprobe 103tcagcatggc gtgtgttggt gagtcctgga aa
3210467DNAArtificialprobe 104tgaccatgag cgacatggtg ctgccggtgc
agacgggagg ttggtctaga ttggatcttg 60ctggcac
6710552DNAArtificialprobe 105gggttcccta agggttggac ggaaagagcc
gaagcatctg taggttcctc ct 5210660DNAArtificialprobe 106tgggtggcag
ggcccagagg aaagtcggcc tggaatgtct agattggatc ttgctggcac
6010776DNAArtificialprobe 107gggttcccta agggttggac aaggaaggcc
tggtttgcct gcagatggat ggtccatcat 60gatctttctt tccagc
7610865DNAArtificialprobe 108gttcttcttg ctgcaggggg cctggccacr
tgagggtaag tgtctagatt ggatcttgct 60ggcac 6510953DNAArtificialprobe
109gggttcccta agggttggac acttctttct gcacaragag kggatctcta agg
5311073DNAArtificialprobe 110acccctcacr cctcgttgga cagatccatg
atggggtctc caaggccart ttctccatcg 60gttccatgat gcg
7311169DNAArtificialprobe 111gggttcccta agggttggac agatatcatg
tttgagcact tctttctgca caaagagtgg 60atctctaaa
6911259DNAArtificialprobe 112tgcccttgca gggacctaca gatgctacgg
ttctggtcta gattggatct tgctggcac 5911361DNAArtificialprobe
113gggttcccta agggttggac cttgggccca gaggaaagtc rgcctggaat
gttccgtkga 60t 6111442DNAArtificialprobe 114gctgcgcact gcagggagcc
tacgttcatg ggcctccccy tc 4211586DNAArtificialprobe 115cctggataga
tggtacatgt cataggagct ccgggagctg caggacaagg tcacattctc 60tcgtctagat
tggatcttgc tggcac 8611657DNAArtificialprobe 116cctggataga
tggagctgca ggacaaggtc acagtctaga ttggatcttg ctggcac
5711749DNAArtificialprobe 117gggttcccta agggttggac taggagaccg
tggaaaaggc aattcccga 4911828DNAArtificialprobe 118cccactggtg
aaatgtggtg ctgatttt 2811957DNAArtificialprobe 119gacactaagt
ggatgaagca gatggatata agcgtctaga ttggatcttg ctggcac
5712069DNAArtificialprobe 120gggttcccta agggttggac ctgaagctcc
tcagctatgg ctctaggatc ataagacatg 60ggacagaca
6912160DNAArtificialprobe 121cgggttttcc tcacctgtga cagaaacaag
cagtgggtca cttgagtttg accacacgca 6012265DNAArtificialprobe
122gggcagggca cggaaagagc cgaagcatct gtagttccct cgtctagatt
ggatcttgct 60ggcac 6512365DNAArtificialprobe 123ggkcayrgca
cggaaagagc cgaagcatct gtagktccct cgtctagatt ggatcttgct 60ggcac
6512445DNAArtificialcontrol probe 124gggttcccta agggttggat
agagggctcc aggttatccg ggctc 4512545DNAArtificialcontrol probe
125gggttcccta agggttggat agagggctcc aggttatccg ggctc
4512652DNAArtificialcontrol probe 126gggttcccta agggttggat
tcaggagtga tacttcacag atcctggagg aa 5212752DNAArtificialcontrol
probe 127aacatcccag tccttaaggc caaactgagt ctagattgga tcttgctggc ac
5212858DNAArtificialcontrol probe 128gggttcccta agggttggac
tgcgtgagac caagcgccgt catgagaccc gactggtg
5812958DNAArtificialcontrol probe 129gagattgaca atgggaagca
gcgtgagttt gagagtctag attggatctt gctggcac
5813044DNAArtificialcontrol probe 130gggttcccta agggttggac
caggcgttct cagcctccgg atga 4413141DNAArtificialcontrol probe
131cacaaatacc agagcacaga ttcaagtgca atccatgtat c
4113245DNAArtificialcontrol probe 132tgtatgggtc attctcacct
ggtctagatt ggatcttgct ggcac 4513360DNAArtificialcontrol probe
133gggttcccta agggttggac gcaggacaga aggagcaagc tgtggaatgg
tataagaaag 6013460DNAArtificialcontrol probe 134gtattgaaga
actggaagaa ggaatagctg ttatagttac aggacaaggt aagattgtat
6013560DNAArtificialcontrol probe 135ttgtttatag ccatcccaaa
ttatgatata ttcacactct agattggatc ttgctggcac
6013658DNAArtificialcontrol probe 136gggttcccta agggttggac
ctgctcccag tagggtcagc atctggaccc caggctga
5813759DNAArtificialcontrol probe 137gagtcaggct ctgattccag
atctagcctc catcatgaag aagctcttga ccaagtatg
5913858DNAArtificialcontrol probe 138acaacctctt tgagacgtcc
tttccctact ccatgtctag attggatctt gctggcac
5813960DNAArtificialcontrol probe 139gggttcccta agggttggac
gcaggacaga aggagcaagc tgtggaatgg tataagaaag
6014060DNAArtificialcontrol probe 140gtattgaaga actggaagaa
ggaatagctg ttatagttac aggacaaggt aagattgtat
6014160DNAArtificialcontrol probe 141ttgtttatag ccatcccaaa
ttatgatata ttcacactct agattggatc ttgctggcac
6014273DNAArtificialcontrol probe 142gggttcccta agggttggac
cagccagcag
cagccccaag ctgataagat taatctaaag 60agcaaattat ggt
7314369DNAArtificialcontrol probe 143gtaatttcct atgctgaaac
tttgtagtta attttttaaa aaggtttcat tttcctattg 60gtctgattt
6914466DNAArtificialcontrol probe 144cacaggaaca ttttacctgt
ttgtgaggca ttttttctcc tggtctagat tggatcttgc 60tggcac
6614573DNAArtificialcontrol probe 145gggttcccta agggttggac
cagccagcag cagccccaag ctgataagat taatctaaag 60agcaaattat ggt
7314669DNAArtificialcontrol probe 146gtaatttcct atgctgaaac
tttgtagtta attttttaaa aaggtttcat tttcctattg 60gtctgattt
6914766DNAArtificialcontrol probe 147cacaggaaca ttttacctgt
ttgtgaggca ttttttctcc tggtctagat tggatcttgc 60tggcac
6614865DNAArtificialcontrol probe 148gggttcccta agggttggac
ccccttaatc caaagtgctg ctcaacaaga gttcgaagac 60aactg
6514969DNAArtificialcontrol probe 149gacaggaatc tcacctttca
taaaatggtg gcatggatga ttgcactcta gattggatct 60tgctggcac
6915057DNAArtificialcontrol probe 150gggttcccta agggttggac
acccttttac actgacatcc gcccctgagg aagactt
5715156DNAArtificialcontrol probe 151ctttagtatc catatccgca
tcgttgggga ctggacagag gggctgttca atgctt 5615257DNAArtificialcontrol
probe 152gtggctgtga taagcaggag tttcaagatg cgtgtctaga ttggatcttg
ctggcac 5715319DNAArtificialshort left probe KIR3DL1 WT
153gcctggttgg acagatcca 1915436DNAArtificiallong left probe KIR3DL1
WT 154ctctaaggac ccctcacgcc tcgttggaca gatcca
3615518DNAArtificialshort left probe KIR3DL1 1*024N 155gcctggttgg
acgatcca 1815635DNAArtificiallong left probe KIR3DL1 1*024N
156ctctaaggac ccctcacgcc tcgttggacg atcca 3515724DNAArtificialshort
right probe KIR3DL1 WT 157cccatgatgc ttgcccttgc agrg
2415844DNAArtificiallong right probe KIR3DL1 WT 158cccatgatgc
ttgcccttgc agrgacctac agatgctacg gttc 4415966DNAArtificial
Sequenceprobe 159gggttcccta agggttggac cgatggcagg gcccagagga
aagtcggcct ggaatgttcc 60gttgat 6616071DNAArtificial Sequenceprobe
160gctgcgcact gcagggagcc tacgttcatg ggcctcccct tcccttggtc
tagattggat 60cttgctggca c 7116166DNAArtificial Sequenceprobe
161gggttcccta agggttggac ctctccagcc aggatgatcc atccccgcac
tccctccctc 60tattcc 6616232DNAArtificial Sequenceprobe
162tttccaggac tcaccaacac acgccatgct ga 3216344DNAArtificial
Sequencelong right probe KIR3DL1 1*024N 163cccatgatgc ttgcccttgc
agggacctac agatgctacg gttc 44
* * * * *
References