U.S. patent application number 13/200991 was filed with the patent office on 2012-05-03 for means and methods for investigating nucleic acid sequences.
Invention is credited to Martin de Boer, Taco Willem Kuijpers.
Application Number | 20120107812 13/200991 |
Document ID | / |
Family ID | 40743878 |
Filed Date | 2012-05-03 |
United States Patent
Application |
20120107812 |
Kind Code |
A1 |
Kuijpers; Taco Willem ; et
al. |
May 3, 2012 |
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. The invention further provides improved calibrators
which are particularly suitable for determining (pseudo)gene
variants and copy number variation.
Inventors: |
Kuijpers; Taco Willem;
(Amstelveen, NL) ; de Boer; Martin; (Blokker,
NL) |
Family ID: |
40743878 |
Appl. No.: |
13/200991 |
Filed: |
October 5, 2011 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
12998595 |
Jul 26, 2011 |
|
|
|
PCT/NL2009/050669 |
Nov 5, 2009 |
|
|
|
13200991 |
|
|
|
|
Current U.S.
Class: |
435/6.11 ;
435/320.1; 536/24.3 |
Current CPC
Class: |
C07H 21/04 20130101;
C12Q 1/6858 20130101; C12Q 1/6883 20130101; C12Q 1/6858 20130101;
C12Q 2525/155 20130101; C12Q 2533/107 20130101; C12Q 2537/143
20130101; C12Q 2533/107 20130101; C12Q 2531/113 20130101; C12Q
1/6858 20130101; C12Q 2600/156 20130101; C12Q 1/6876 20130101; C12Q
2537/143 20130101 |
Class at
Publication: |
435/6.11 ;
536/24.3; 435/320.1 |
International
Class: |
C12Q 1/68 20060101
C12Q001/68; C12N 15/63 20060101 C12N015/63; C07H 21/04 20060101
C07H021/04 |
Foreign Application Data
Date |
Code |
Application Number |
Nov 5, 2008 |
NL |
PCT/NL2008/050698 |
Claims
1. A nucleic acid molecule comprising a sequence which has at least
70% sequence identity with at least one nucleic acid sequence
consisting of: a) a probe set of FIGS. 3A, 3B, 3C, 3D, 3F and/or
3G, without the primer binding sites GGGTTCCCTAAGGGTTGGA and
TCTAGATTGGATCTTGCTGGCAC and TCTAGATTGGATCTTGCTGGCGC of said probe
sets, or b) a complementary sequence of said probe set without said
primer binding sites, wherein said nucleic acid sequence of a) or
b) either comprises immediately adjacent to each other: the
sequences or complementary sequences of a left probe of said probe
set, without primer binding site GGGTTCCCTAAGGGTTGGA, and of a
right probe of the same probe set, without primer binding site
TCTAGATTGGATCTTGCTGGCAC or TCTAGATTGGATCTTGCTGGCGC, if said probe
set consists of two probes; or the sequences or complementary
sequences of a left probe of said probe set, without primer binding
site GGGTTCCCTAAGGGTTGGA, and of a middle probe, and right probe of
the same probe set, without primer binding site
TCTAGATTGGATCTTGCTGGCAC or TCTAGATTGGATCTTGCTGGCGC, if said probe
set consists of three probes.
2. A nucleic acid molecule according to claim 1, further comprising
at least one control nucleic acid sequence.
3. A nucleic acid molecule according to claim 1, comprising at
least five nucleic acid sequences as defined in claim 1.
4. A nucleic acid molecule according to claim 1, comprising
sequences selected from the group consisting of: all nucleic acid
sequences of FIG. 3A without the primer binding sites
GGGTTCCCTAAGGGTTGGA and TCTAGATTGGATCTTGCTGGCAC and
TCTAGATTGGATCTTGCTGGCGC, or sequences having at least 70% sequence
identity to said nucleic acid sequences, and all nucleic acid
sequences of FIG. 3B without the primer binding sites
GGGTTCCCTAAGGGTTGGA and TCTAGATTGGATCTTGCTGGCAC and
TCTAGATTGGATCTTGCTGGCGC, or sequences having at least 70% sequence
identity to said nucleic acid sequences, and all nucleic acid
sequences of FIG. 3C without the primer binding sites
GGGTTCCCTAAGGGTTGGA and TCTAGATTGGATCTTGCTGGCAC and
TCTAGATTGGATCTTGCTGGCGC, or sequences having at least 70% sequence
identity to said nucleic acid sequences, and all nucleic acid
sequences of FIG. 3D without the primer binding sites
GGGTTCCCTAAGGGTTGGA and TCTAGATTGGATCTTGCTGGCAC and
TCTAGATTGGATCTTGCTGGCGC, or sequences having at least 70% sequence
identity to said nucleic acid sequences, and all nucleic acid
sequences of FIG. 3F without the primer binding sites
GGGTTCCCTAAGGGTTGGA and TCTAGATTGGATCTTGCTGGCAC and
TCTAGATTGGATCTTGCTGGCGC, or sequences having at least 70% sequence
identity to said nucleic acid sequences, and all nucleic acid
sequences of FIG. 3G without the primer binding sites
GGGTTCCCTAAGGGTTGGA and TCTAGATTGGATCTTGCTGGCAC and
TCTAGATTGGATCTTGCTGGCGC, or sequences having at least 70% sequence
identity to said nucleic acid sequences, and any combination
thereof, and any complementary sequences thereof.
5. A nucleic acid molecule according to claim 1, comprising at
least two, preferably at least five, control nucleic acid
sequences.
6. A nucleic acid molecule according to claim 2, wherein said
control nucleic acid sequence comprises a sequence of at least 10
nucleotides, preferably at least 20 nucleotides, which has at least
70% sequence identity with a sequence of a gene which has a
constant copy number in the human genome.
7. A nucleic acid molecule according to claim 2, wherein said at
least one control nucleic acid sequence comprises a sequence of at
least 10 nucleotides, preferably at least 20 nucleotides, which has
at least 70% sequence identity with a sequence of a gene encoding a
protein selected from the group consisting of FGF3, BCAS4, LMNA,
GALT, SPG4, IL-4 and NF2, or any combination thereof, or with a
complementary sequence thereof.
8. A nucleic acid molecule according to claim 2, wherein said at
least one control nucleic acid comprises a sequence of at least 10
nucleotides, preferably at least 20 nucleotides, which has at least
70% sequence identity with at least one nucleic acid sequence
consisting of: a) a probe set of FIGS. 3E and/or 3H, without the
primer binding sites GGGTTCCCTAAGGGTTGGA and
TCTAGATTGGATCTTGCTGGCAC and TCTAGATTGGATCTTGCTGGCGC of said probe
sets, or b) a complementary sequence of said probe set without said
primer binding sites, wherein said nucleic acid sequence of a) or
b) either comprises immediately adjacent to each other: the
sequences or complementary sequences of a left probe of said probe
set, without primer binding site GGGTTCCCTAAGGGTTGGA, and of a
right probe of the same probe set, without primer binding site
TCTAGATTGGATCTTGCTGGCAC or TCTAGATTGGATCTTGCTGGCGC, if said probe
set consists of two probes; or the sequences or complementary
sequences of a left probe of said probe set, without primer binding
site GGGTTCCCTAAGGGTTGGA, and of a middle probe and of a right
probe of the same probe set, without primer binding site
TCTAGATTGGATCTTGCTGGCAC or TCTAGATTGGATCTTGCTGGCGC, if said probe
set consists of three probes.
9. A nucleic acid molecule according to claim 1, wherein at least
one of said nucleic acid sequences or complementary sequences
thereof and/or at least one of said control nucleic acid sequences
or complementary sequences thereof are followed from 5' to 3' by a
non-coding sequence of at least 5, preferably at least 20
nucleotides.
10. A nucleic acid molecule comprising a sequence which has at
least 70% sequence identity with a sequence as depicted in FIG.
19.
11. A vehicle or plasmid comprising at least one nucleic acid
molecule according to claim 1.
12. A method for determining the copy number of at least one KIR
gene of an individual comprising: amplifying a sequence with a
length of at least 10 nucleotides of said at least one KIR gene
using a sample of said individual and amplifying a sequence with a
length of at least 10 nucleotides of said at least one KIR gene
using a reference sample, said reference sample comprising a
nucleic acid molecule according to claim 1 or a plasmid; and
amplifying a sequence with a length of at least 10 nucleotides of
at least one control gene using said sample of said individual and
amplifying a sequence with a length of at least 10 nucleotides of
said at least one control gene using said reference sample;
determining a level of amplified product of said sequence of said
at least one KIR gene from said sample of said individual and
determining a level of amplified product of said sequence of said
at least one KIR gene from said reference sample; and determining a
level of amplified product of said sequence of said at least one
control gene from said sample of said individual and determining a
level of amplified product of said sequence of said at least one
control gene in said reference sample; and comparing said levels of
amplified products of said sequences of said at least one KIR gene
with each other and with said levels of amplified products of said
sequences of said at least one control gene, thereby determining
the copy number of said at least one KIR gene.
13. A method according to claim 12, further comprising the steps
of: a) adding to said sample of said individual and to said
reference sample at least one probe set selected from FIGS. 3A, 3B,
3C, 3D, 3F and/or 3G, and b) optionally, adding to said sample of
said individual and to said reference sample at least one probe set
selected from FIG. 3E or 3H, and c) allowing hybridization of said
probe set or probe sets to complementary nucleic acid of said
sample of said individual, and d) allowing hybridization of said
probe set or probe sets to complementary nucleic acid of said
reference sample, and e) subjecting nucleic acid of said sample of
said individual, and nucleic acid of said reference sample to a
ligation reaction.
14. A method according to claim 13, further comprising amplifying
ligated nucleic acid and determining levels of amplified products,
thereby determining the copy number of at least one KIR gene of
said individual.
15. A method according to claim 12, wherein at least one of said
probe sets selected from FIGS. 3A, 3B, 3C, 3D, 3F and/or 3G
comprises a third nucleic acid probe.
16. A method for determining a KIR haplotype of an individual
comprising determining the copy number of at least 5, preferably at
least 10, more preferably at least 15, most preferably all KIR
genes of said individual with a method according to claim 12.
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, 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 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 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,
[0026] b) allowing hybridization of said at least two different
probe sets to complementary nucleic acid of said sample,
[0027] c) subjecting nucleic acid of said sample to a ligation
reaction, and
[0028] 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.
[0029] 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.
[0030] 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.
[0031] 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.
[0032] 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:
[0033] a) adding to said sample at least two different probe sets,
each probe set comprising: [0034] 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 [0035] 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,
[0036] 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
[0037] 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
[0038] 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,
[0039] b) allowing hybridization of said at least two different
probe sets to complementary nucleic acid of said sample,
[0040] c) subjecting nucleic acid of said sample to a ligation
reaction,
[0041] 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
[0042] e) determining whether amplified nucleic acid is present,
thereby determining whether said at least one target nucleic acid
sequence is present in said sample,
[0043] wherein at least one third nucleic acid probe is
complementary to a target nucleic acid region comprising a
(pseudo)gene variation.
[0044] 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.
[0045] 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).
[0046] 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.
[0047] 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.
[0048] 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.
[0049] 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.
[0050] 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.
[0051] 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.
[0052] 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.
[0053] 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.
[0054] 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.
[0055] 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:
[0056] a first nucleic acid probe, said first probe comprising
[0057] 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 [0058] a second nucleic acid probe,
said second probe comprising [0059] 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,
[0060] 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
[0061] 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
[0062] 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
[0063] 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.
[0064] 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.
[0065] 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).
[0066] 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 provide KIR-specific probes which
provide particularly good results. These probes are therefore
preferred when a KIR locus is investigated. FIGS. 3C and D provide
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.
FIGS. 3F and G also provide preferred probes. 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, 3D, 3F or 3G, is used.
Preferably, at least one probe depicted in FIG. 3C, 3D, 3F or 3G is
used. More 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, 3D, 3F
or 3G, preferably depicted in FIG. 3C, 3D, 3F or 3G, are used.
[0067] 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. Also provided
is a method and/or a use according to the invention, wherein at
least one probe set depicted in FIG. 3F selected from the group
consisting of probe set 415, 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. 3G 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, probe set 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.
[0068] 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 506, probe set 417, probe set 517, probe
set 409, probe set 507, probe set 710, probe set 709, probe set
708, probe set 704 and probe set 538 of FIG. 3A, probe set 415,
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 of FIG. 3B, probe set 506, probe set 417, probe
set 517, probe set 409, probe set 507, probe set 710, probe set
709, probe set 708, probe set 704 and probe set 538 of FIG. 3C,
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 of FIG. 3D,
probe set 409, probe set 506, probe set 507, probe set 538, probe
set 417 and probe set 517 of FIG. 3F and probe set 408, probe set
507, probe set 528, probe set 413, probe set 416 and probe set 415
of FIG. 3G, 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 FIGS. 3A, and/or 3B, and/or
3C, and/or 3D and/or 3F and/or 3G are used. In a preferred
embodiment all probe sets depicted in FIG. 3C and/or FIG. 3D and/or
FIG. 3F and/or FIG. 3G are used.
[0069] 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).
[0070] The left and right probes of the probe sets of FIG. 3
contain a primer binding site, which are indicated in bold and/or
underlined in this figure. The primer binding site of each left
probe of FIG. 3 consists of the sequence GGGTTCCCTAAGGGTTGGA and
the primer binding site of each right probe of FIG. 3 consists of
the sequence TCTAGATTGGATCTTGCTGGCAC or
TCTAGATTGGATCTTGCTGGCAC.
[0071] Of course, these primer binding sites can be varied at will,
as long as complementary primers are used in the amplification
reaction. Therefore, the primer binding sites of probes according
to the invention need not be at least 70% identical to the above
mentioned sequences. Lower sequence identity can be used,
complementary to the amplification primers. Other primers then used
in the Examples can be developed for use in a method according to
the invention wherein probes depicted in FIG. 3 are used. This
means that the primer binding sites of probes depicted in FIG. 3
can be amended or even replaced by entirely different primer
binding sites. Thus, the sequence identity between a primer binding
site of a probe according to the invention and a primer binding
site as depicted in FIG. 3 can be less than 70%, such as for
instance at most 60%, at most 50%, at most 40%, at most 30%, at
most 25%, at most 20%, at most 15%, at most 10% or even lower.
[0072] However, the parts of probes according to the invention that
are capable of hybridizing to KIR genes preferably have at least
70% sequence identity to the KIR-specific sequences depicted FIG.
3, i.e. the probe sequences of FIG. 3 without the primer binding
sites. Thus, preferably at least one probe is used in a method
according to the invention 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 the part of
the sequence of a probe depicted in FIG. 3 which is capable of
hybridizing to a KIR gene, i.e. the part of a sequence of a probe
depicted in FIG. 3 which is not the primer binding site.
[0073] Preferably, probe sets are used which are based on the probe
sets depicted in FIG. 3A, 3B, 3C, 3D, 3F or 3G, preferably based on
the probe sets depicted in FIGS. 3C and/or 3D and/or 3F and/or 3G.
At least one of said probe sets 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 without the primer binding sites. This means
that the probes of said probe set have at least 70% sequence
identity to the KIR-specific parts of 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 506, probe set 417, probe
set 517, probe set 409, probe set 507, probe set 710, probe set
709, probe set 708, probe set 704 and probe set 538 of FIG. 3A,
probe set 415, 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 of FIG. 3B, probe set 506,
probe set 417, probe set 517, probe set 409, probe set 507, probe
set 710, probe set 709, probe set 708, probe set 704 and probe set
538 of FIG. 3C, 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
of FIG. 3D, probe set 409, probe set 506, probe set 507, probe set
538, probe set 417 and probe set 517 of FIG. 3F and probe set 408,
probe set 507, probe set 528, probe set 413, probe set 416 and
probe set 415 of FIG. 3G, without the primer binding sequences,
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 FIGS. 3A, B, C, D, F and G, as described
above. Further provided are therefore probes and probe sets as
depicted in FIG. 3A, 3B, 3C, 3D, 3F or 3G, 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 the KIR-specific part
of a probe or probe set depicted in FIG. 3A, 3B, 3C, 3D, 3F or 3G,
i.e. the sequences depicted in FIG. 3 without the primer binding
sites. 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, 3D, 3F or 3G
without the primer binding sites. 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, 3D, 3F or 3G.
[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
TCTAGATTGGATCTTGCTGGCGC-3', or the complements thereof. These
primers are particularly suitable for amplifying probe sets
depicted in FIG. 3. However, as described above, different primers
are also suitable, in which case the primer binding sites need to
be altered in order to be complementary to the primers.
[0076] The invention further provides calibrators that are
particularly suitable for determining copy numbers of nucleic acids
of interest. Currently only relative gene copy number can be
determined. This is often done by graphically representing
amplified products of genes of interest by separate peaks. A
relative peak intensity (height or surface) of an amplified product
of a gene of interest is compared with the peak intensity of an
amplified product of a control sample containing the gene of
interest to determine relative copy number. For instance, if an
MLPA reaction is used, each peak represents the product of an
amplified MLPA ligated probe. However, with such a method it is not
possible to quantify the absolute gene copy number because
intensity peaks of a control sample do not represent a known copy
number. Furthermore, a reference sample does not always contain all
genes of interest. This is in particular the case for polygenic and
highly polymorphic gene loci such as the KIR locus and the human
leukocyte antigen (HLA) locus, whereby the identity and copy number
of alleles differ greatly between individuals. Since no individual
has all alleles of such polygenic and highly polymorphic gene
cluster, a reference sample containing all these alleles is not
available. Thus, if a sample of a random individual is compared
with such a reference sample in order to determine the haplotype
and/or copy number of genes, possibly several alleles of said
individual are not detected because they are not present in the
reference sample. Thus, with a reference sample currently used in
the art, it is not possible to determine the complete haplotype
(including copy number variation) of such polygenic and highly
polymorphic gene loci such as the KIR locus of an individual. It is
of course possible to use multiple reference samples, which will
result in a more elaborate method. Furthermore, some alleles of a
gene cluster are relatively rare and it is difficult to obtain
reference samples with all known alleles of a gene cluster.
[0077] The invention provides means and methods that enable
determination of the complete haplotype of a polygenic and highly
polymorphic gene cluster. In addition, determination of copy number
variation of genes of such gene cluster of an individual has now
become possible. This comprises the use of a nucleic acid molecule
comprising at least one control nucleic acid sequence, and for each
gene or allele of interest a nucleic acid sequence which is unique
for said gene or allele of interest. Said nucleic acid molecule can
be used as such. Of course, such nucleic acid molecule can also be
present in a vehicle such as for instance a plasmid, which
optionally comprises other nucleic acid sequences. Such nucleic
acid molecule as such or a vehicle or plasmid comprising such
nucleic acid molecule are herein referred to as a calibrator
according to the invention. Instead of a single nucleic acid
molecule, a calibrator according to the invention can also contain
a combination of multiple nucleic acid molecules or multiple
vehicles/plasmids according to the invention. For instance, a
calibrator according to the invention may contain 2, 3, 4, 5, 6, 7,
8 or more separate nucleic acid molecules. Preferably, however, a
calibrator according to the invention consists of one nucleic acid
molecule, vehicle or plasmid. In FIG. 17 an exemplary
representation of such vehicle is shown which comprises multiple
nucleic acid sequences unique for a gene or allele of interest, KIR
genes in FIG. 17, to which, for instance, MLPA probes can bind.
[0078] A calibrator according to the invention comprises at least
one nucleic acid sequence with a length of at least 10 nucleotides
which is at least 70% identical to a part of a (pseudo)gene
comprising a polymorphism, such as a SNP. A part of a (pseudo)gene
is defined as a consecutive stretch of at least 10 nucleotides in
said (pseudo)gene, Preferably said part is at least 15, more
preferably at least 20, more preferably at least 25, more
preferably at least 30 nucleotides, such as 35, 40, 45 or 50
nucleotides. Said nucleic acid sequence of a calibrator according
to the invention preferably comprises a sequence which is identical
to--or complementary to--a polymorphism of said (pseudo)gene.
Preferably, a calibrator according to the invention comprises at
least one nucleic acid sequence which is at least 75%, more
preferably at least 80%, more preferably at least 85%, more
preferably at least 90%, more preferably at least 95% identical to
part of a gene, which part comprises at least one polymorphism.
[0079] In addition, such calibrator comprises at least one nucleic
acid sequence which is at least 70% identical to part of a control
gene. A part of a control gene is defined as a consecutive stretch
of at least 10 nucleotides in said control gene, preferably at
least 15, more preferably at least 20, more preferably at least 25,
more preferably at least 30 nucleotides, such as 35, 40, 45 or 50
nucleotides. As used herein, control genes are preferably genes
which have a constant copy number in the human genome. Preferably a
calibrator according to the invention comprises at least one
nucleic acid sequence which is at least 75%, more preferably at
least 80%, more preferably at least 85%, more preferably at least
90%, more preferably at least 95% identical to part of a control
gene. Preferably sequences are used of control genes that have no
or few polymorphisms so that these sequences will always be present
in samples of individuals, avoiding the need to use many different
control sequences for one particular control gene.
[0080] In one embodiment, a calibrator according to the invention
comprises a nucleic acid sequence which is identical to part of a
gene, which part comprises at least one polymorphism and a nucleic
acid sequence which is identical to part of a control gene. As
described before, such part contains at least 10 nucleotides. In
one embodiment, a calibrator according to the invention comprises
at least one nucleic acid sequence which is at least 70% identical
to part of a gene, which part comprises at least two polymorphisms,
such as two SNPs.
[0081] A calibrator is preferably designed such that all genes of
interest and their allelic variants are separately represented once
with a unique nucleic acid sequence on the calibrator. Different
unique nucleic acid sequences from one gene of interest or gene
variant of interest may also be represented on a calibrator as a
single sequence to yield the same result: i.e. detection by one
single probe in a mixture of probes as one copy of that sequence on
the calibrator used.
[0082] With the use of a calibrator according to the invention, it
is possible to determine in one reaction polymorphisms as well as
absolute copy numbers of (pseudo)genes. In a preferred embodiment,
nucleic acid sequences representing all possible (pseudo)genes of a
gene cluster are present on a calibrator according to the
invention. As used herein, "representing all possible (pseudo)genes
of a gene cluster" means that for each (pseudo)gene of a gene
cluster a nucleic acid sequence which is at least 70% identical to
a part of at least 10 nucleotides of said (pseudo)gene is present
on the calibrator. Said part of said (pseudo)gene preferably
comprises at least one polymorphism, such as a SNP. Such calibrator
according to the invention comprising nucleic acid sequences
representing all possible (pseudo)genes of a gene cluster allows
for the determination of presence or absence, as well as the copy
number, of each of said (pseudo)genes in an individual, using a
sample of said individual. Thus, such calibrator enables
determining the entire haplotype of a polygenic and polymorphic
gene cluster of an individual.
[0083] Now that a calibrator according to the invention is provided
by the invention, an improved reference sample when determining
(absolute) copy number variation has become available. This is for
instance shown in Example 5 for determining the presence and copy
number of KIR genes, which form a particularly polygenic and highly
polymorphic gene locus. A calibrator according to the invention can
be advantageously used with any method known to a person skilled in
the art for detecting nucleic acid, such as (real-time) PCR,
PCR-SSP, multiplex PCR and PCR-SSOP and MLPA. However, a calibrator
according to the invention is particularly suitable for use in an
MLPA method according to the invention.
[0084] An example of how a calibrator according to the invention
can be used in a reference sample in an MLPA reaction is as
follows. A test sample with nucleic acid of an individual and a
reference sample comprising a calibrator according to the invention
are provided with MLPA probes. Following ligation of MLPA probes to
nucleic acid of said test sample and to said calibrator of said
reference sample, an amplification reaction with both the reference
sample with calibrator and the test sample is performed. A
calibrator according to the invention and the MLPA probes are
designed such that each amplified nucleic acid variant of a
(pseudo)gene containing a polymorphism has a different length. It
is thus immediately apparent which variant of a (pseudo)gene is
present from the presence and length of amplified product.
Furthermore, the amount of amplification products derived from
(pseudo)genes of interest of the test sample can be correlated to
the amount of amplification products of the calibrator of the
reference sample. Also, the amount of amplification product derived
from a control gene of the test sample, of which the copy number in
the genome is known, can be correlated to the amount of
amplification product of the corresponding control sequence of the
calibrator of the reference sample. Based on the correlations of
the amounts of amplified product of a (pseudo)gene of interest
between the test sample and the reference sample, and the
correlations of the amounts of amplified product of a control gene
(with a constant copy number in the human genome) between the test
sample and the reference sample, the copy number of the
(pseudo)genes of interest in an individual can be determined. A
more detailed example of the determination of the copy number of a
gene, when used with a calibrator according to the invention in an
MLPA method, is described below.
[0085] An MLPA method according to the invention comprises the use
of a sample with nucleic acids obtained from an individual ("a test
sample") and a reference sample comprising a calibrator according
to the invention ("a reference sample"). In one embodiment, both
said test sample and said reference sample containing the
calibrator are subjected to an MLPA method, preferably an MLPA
method according to the invention. This comprises the addition to
said test sample and said reference sample of at least one probe
set which is complementary to part of a (pseudo)gene of interest
(said part preferably comprising a polymorphism), and at least one
probeset which is complementary to part of a control gene. The
probe sets are allowed to hybridize to the target nucleic acid in
said test sample and to the target nucleic acid located on the
calibrator in the reference sample. Said target nucleic acid
located on the calibrator is at least 70% identical to target
nucleic acid in said test sample. Subsequently the probes of the
different probe sets hybridized to nucleic acid in said test sample
and in said reference sample are subjected to a ligation reaction.
As herein before described in detail, ligation will only occur if
probes of a specific probeset are hybridized immediately adjacent
to each other on their target sequences. Thus, if one specific
variant of a polymorphic gene is present in the test sample, only
the probes of the probe set specific for this specific variant will
ligate, whereas the probes of probe sets specific for other gene
variants will not ligate. As described before, a ligated probe set
according to the invention is flanked by two primer binding sites.
During a subsequent amplification reaction, only the ligated probe
sets will be amplified. Hence, the presence or absence of each gene
variant in a test sample is directly determined.
[0086] The calibrator, however, preferably contains binding sites
for each of the probe sets, so that amplification of all probe
sequences will occur in the reference sample. This avoids
false-negative test results; if a given probe sequence is not
amplified in the test sample, it is verified whether the
corresponding probe sequence is amplified in the reference sample,
i.e. from the calibrator. Only if the probe sequence is indeed
amplified in the reference sample, the absence of the probe
sequence in the test sample is considered a reliable result. If the
probe sequence appears not to be amplified in the reference sample
either, this indicates a failure of the test procedure and the test
results are to be discarded. Hence, false-negative test results are
avoided. A binding site for a particular probe set is a nucleic
acid sequence that is at least 70% identical to part of a
(pseudo)gene to which said probe set is complementary. As used
herein, reference to a gene of interest also encompasses a
pseudogene of interest.
[0087] Besides probe sets specific for a gene of interest, probe
sets specific for control genes are used. Control genes preferably
have a constant copy number in the human genome, such as for
instance two. This copy number is known. Nucleic acid sequences
with a length of at least 10 nucleotides which are at least 70%
identical to control gene sequences and nucleic acid sequences with
a length of at least 10 nucleotides which are at least 70%
identical to gene of interest sequences are present in known
amounts on the calibrator. It is therefore possible to correlate
amplified products from the calibrator to amplified products of a
test sample. This is for instance done as follows. Amplification
reactions of the test sample and of the reference sample will
result in an intensity peak pattern with peaks for each amplified
nucleic acid product. The peaks will have varying intensity (for
instance height or surface). The peaks represent amplified nucleic
acid sequences indicative for a gene of interest or a control gene.
The peak intensity (for instance height or surface) of an amplified
control gene product of the test sample and the peak intensity of
the amplified product of the same control gene sequence in the
reference sample (i.e. of the calibrator) are compared. This is
done for each control gene sequence. The peak intensities of both
amplified products ought to be the same, representing for instance
2 copies per genome or DNA sample tested if the copy number of the
control gene is 2. The control genes are also an internal quality
control of both the test and the reference sample, because
amplified product is only detected if the MLPA reaction was
successful. The proportion between the peak intensities of
amplified control gene product in the test sample and in the
reference sample can be determined based on relative differences in
peak intensity. Subsequently, the peak intensity of amplified
product of a gene of interest of the test sample and the peak
intensity of amplified product of a sequence of the same gene of
the reference sample (i.e. of the calibrator) are also compared.
This is also done for each gene of interest. The proportion between
the peak intensities of amplified product of each gene of interest
in the test sample and in the reference sample is determined based
on relative differences in peak intensity as well.
[0088] If nucleic acid corresponding to a specific nucleic acid
sequence in a control gene and nucleic acid corresponding to a to a
specific nucleic acid sequence in a gene of interest are present on
the calibrator in the same amount, equal amounts of product will in
principle be amplified during the amplification reaction. In that
case, the peak intensities of the control gene and the gene of
interest can be directly compared. If the control gene and the gene
of interest are present in the same amount in the test sample as
well, the difference between the peak intensities of control gene
in test sample and reference sample will be comparable to the
difference between the peak intensities of the gene of interest in
the test sample and reference sample. Thus, the proportion between
the peak intensities of amplified product of the gene of interest
in the test and reference sample, and the proportion between the
peak intensities of amplified control gene product in the test and
reference sample are equal if the copy number of the gene of
interest and the copy number of the control gene are equal. For
instance, if the copy number in the human genome of a control gene
is 2, and if the peak intensity of amplified control gene product
in a reference sample is determined to be 2 and the relative peak
intensity of amplified control gene product in a test sample is
determined to be 3, the proportion between the peak intensities of
amplified product of the control gene in the test sample versus the
reference sample is 3/2=1.5. If it is determined that the
proportion between the peak intensities of amplified product of a
gene of interest in the test sample versus the reference sample is
also 1.5, it can be concluded that the copy number of the gene of
interest is identical to the copy number of said control gene, that
is 2. This direct comparison of peak intensity proportions only
applies if nucleic acid corresponding to part of the control gene
and nucleic acid corresponding to part of the gene of interest are
present in the same amount on the calibrator. It is, therefore,
preferred to use the same number of different nucleic acid
sequences on the calibrator. Otherwise, the difference between the
number of different nucleic acid sequences on the calibrator needs
to be taken into account when calculating the copy number of the
corresponding genes in the test sample.
[0089] When equal numbers of different nucleic acid sequences are
present on the calibrator, the proportion between the peak
intensities of amplified product of a gene of interest in the test
and reference sample, and the proportion between the peak
intensities of amplified control gene product in the test and
reference sample, are substantially equal if the copy number of the
control gene and the copy number of the gene of interest in a test
sample are the same, and these proportions are not equal if the
copy number of the control gene and the copy number of the gene of
interest in a test sample are different. The copy number of the
gene of interest in the test sample can then be calculated based on
the peak intensity proportions of the control gene of the test
sample and the control gene of the reference sample and the peak
intensity proportions of the gene of interest of the test sample
and the gene of interest of the reference sample, making use of the
known copy number in the test sample of the control gene. For
instance, if, like in the example above, the copy number of a
control gene is 2, and if the peak intensity of amplified control
gene product in the reference sample is determined to be 2 and the
relative peak intensity of amplified control gene product in the
test sample is determined to be 3, the proportion between the peak
intensity of amplified product of the control gene in the test
sample versus the reference sample is 3/2=1.5. If the proportion
between the peak intensities of amplified product of a gene of
interest in test and reference sample is determined to be 3,
instead of 1.5, it can be concluded that the copy number of the
gene of interest is twice (3/1.5) the copy number of the control
gene, that is 4. It is also possible to determine the proportion of
the peak intensity of the control gene and the peak intensity of
the gene of interest of the reference sample and to compare this
with the proportion of the peak intensities of amplification
product of the corresponding genes of the reference sample. If this
proportion of the reference sample is about one (meaning that the
peak intensities are about the same, which is often the case when
the number of gene-specific sequences and control-specific
sequences on the calibrator are the same), and if this proportion
of the test sample is 2, it can be concluded that the copy number
of the gene of interest in the individual is twice the (known) copy
number of the control gene in the individual.
[0090] Although not necessary, before determining the copy number
of a gene of interest in a test sample, the concentration of total
nucleic acid in the test sample is preferably measured, for
instance using spectrometry. The molecular weight of a calibrator
according to the invention is, of course, also known. The
concentration of nucleic acid in the test sample and the
concentration of nucleic acid (i.e. calibrator) in a reference
sample can then be made approximately equal, so that the peak
intensities of amplified product of control genes, which have a
constant copy number, of the test sample and of the reference
sample are approximately equal. In that case, a direct comparison
between the peak intensities of amplified product of a gene of
interest of a test sample and a reference sample can be made. Then,
the differences in nucleic acid concentration in the test and
references samples do not need to be taken into account.
[0091] For instance, the concentration of nucleic acid in the test
sample and the concentration of the calibrator in the reference
sample can both be based on the molecular weight of the human
genome. If, for example, the amount of genomic DNA added in a test
sample of the assay is 100 nanogram, the concentration of genomic
DNA in the assay is then 4.8E-15 Mol/liter, based on the fact that
a diploid human female and male nuclei in G.sub.1 phase of the cell
cycle should contain 6.950 and 6.829 pg of DNA, respectively. It is
then possible to prepare a reference sample with calibrator in the
same concentration as the concentration of nucleic acid in the test
sample, because the weight of the calibrator can be calculated if
the exact composition of the calibrator is known. In that case, a
copy number of for instance 2 for a control gene in the test sample
will result in the same amount of amplified product in both the
test and the reference sample. It follows that the peak intensities
of this control gene product will then be approximately equal for
the test sample and the reference sample.
[0092] Preferably, a calibrator according to the invention
comprises the same number of copies of each nucleic acid sequence.
For instance, 1 copy of each nucleic acid which is at least 70%
identical to part of a gene of interest and 1 copy of each nucleic
acid which is at least 70% identical to part of a control gene are
preferably present on the calibrator.
[0093] Accordingly, the invention provides a nucleic acid molecule
comprising at least one control nucleic acid sequence and at least
one nucleic acid sequence with a length of at least 10 nucleotides
which is at least 70% identical to part of a gene or pseudogene of
interest, or a complementary sequence thereof, wherein at least
80%, preferably at least 85%, more preferably at least 90%, more
preferably at least 95%, of said nucleic acid sequences which are
at least 70% identical to part of at least one gene or pseudogene
of interest comprise a sequence that is identical to, or
complementary to, a gene-specific nucleotide and/or a pseudo-gene
specific nucleotide and/or a gene-specific sequence and/or a
pseudogene-specific sequence and/or an additional polymorphism
within said gene or pseudogene, said polymorphism preferably
comprising an SNP. Preferably, a nucleic acid molecule according to
the invention comprises at least one nucleic acid sequence with a
length of at least 10 nucleotides which is at least 75%, more
preferably at least 80%, more preferably at least 85%, more
preferably at least 90%, more preferably at least 95% identical to
a part of a gene or pseudogene of interest, or a complementary
sequence thereof, said part comprising a (pseudo)gene-specific
nucleotide and/or sequence, and/or an additional
(pseudo)gene-specific polymorphism preferably an SNP. In one
embodiment, a nucleic acid molecule according to the invention
comprises at least one nucleic acid sequence with a length of at
least 10 nucleotides which is identical to a part of a gene or
pseudogene of interest, or a complementary sequence thereof, said
part comprising a (pseudo)gene-specific nucleotide and/or sequence,
and/or an additional (pseudo)gene-specific polymorphism preferably
an SNP. The invention further provides a vehicle or plasmid
comprising a nucleic acid molecule according to the invention.
[0094] As used herein, a "nucleic acid molecule" or a "nucleic acid
sequence" comprises a chain of nucleotides, preferably DNA and/or
RNA. A nucleic acid molecule or nucleic acid sequence of the
invention may be single stranded or double stranded. In other
embodiments a nucleic acid molecule or nucleic acid sequence of the
invention comprises other kinds of nucleic acid structures such as
for instance a DNA/RNA helix, peptide nucleic acid (PNA), locked
nucleic acid (LNA) and/or a ribozyme. Hence, the term "nucleic acid
sequence" also encompasses a chain comprising non-natural
nucleotides, modified nucleotides and/or non-nucleotide building
blocks which exhibit the same function as natural nucleotides.
[0095] As used herein, "copy number of a (control) gene or
pseudogene" refers to the number of DNA molecules of said gene or
pseudogene in the genome of an individual.
[0096] The term "complementary" is known in the art. A
complementary sequence as used herein refers to a nucleic acid
sequence of which the base pairs can be non-covalently connected to
the target sequence.
[0097] As used herein, a "vehicle" is defined as any means that can
contain a nucleic acid molecule, such as for instance a vector or
plasmid. A "plasmid" is defined herein as a circular,
double-stranded DNA molecule.
[0098] As used herein, the term "% sequence identity to part of a
gene" is defined as the percentage of residues in a nucleotide
sequence that is identical with the residues in said part of a gene
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).
[0099] As used herein a "control nucleic acid sequence" is a
nucleic acid sequence with a length of at least 10 nucleotides
which is at least 70% identical to part of a gene other than the
gene of interest, or a complementary sequence thereof. Preferably,
a control nucleic acid sequence is at least 75%, more preferably at
least 80%, more preferably at least 85%, more preferably at least
90%, more preferably at least 95% identical to part of a gene other
than the gene of interest, or a complementary sequence thereof.
Preferably, gene other than the gene of interest, herein called a
control gene, has a constant copy number in the human genome. Most
preferably, it is known how many copies of said control gene are
present in the genome of each human. Said control gene is thus
preferably not subject to copy number variation. In a preferred
embodiment, said control gene has two copies in the human genome.
The invention therefore also provides a nucleic acid molecule
according to the invention, wherein said control nucleic acid
sequence is at least 70% identical to, or complementary to, a part
of a control gene which has a constant copy number in the human
genome, preferably wherein said control gene has a copy number of
two in the human genome.
[0100] Examples of genes which are normally not subject to copy
number variation and which are known to have a copy number of 2 are
FGF3, BCAS4, LMNA, PARK2, MSH6, GALT, SPG4, IL-4 and NF2.
Therefore, in a preferred embodiment, said at least one control
nucleotide sequence is at least 70% identical to, or complementary
to, a part of FGF3, BCAS4, LMNA, PARK2, MSH6, GALT, SPG4, IL-4
and/or NF2, said part having a length of at least 10 nucleotides.
In FIGS. 3E and 3H, probe sets that are particularly suitable to
hybridize to these control genes are depicted. In a preferred
embodiment, said at least one control nucleotide sequence therefore
has at least 70% sequence identity with a probe of at least one
probe set of FIGS. 3E and/or 3H, or complementary sequences of said
probes. More preferably said at least one control nucleotide
sequence has at least 80%, more preferably at least 85%, more
preferably at least 90%, most preferably at least 95% sequence
identity with a probe of at least one probe set of FIGS. 3E and/or
3H, or complementary sequences of said probes. Preferably, control
nucleotide sequences are used which together have at least 70%,
more preferably at least 80%, more preferably at least 85%, more
preferably at least 90%, most preferably at least 95% sequence
identity with all probes of at least one probe set of FIGS. 3E
and/or 3H, or complementary sequences thereof. In one embodiment,
control nucleotide sequence are used that together are identical to
the sequence of all probes of at least one probe set of FIGS. 3E
and/or 3H, or complementary sequences thereof.
[0101] The left and right probes of the probesets of FIGS. 3E and
3H contain a primer binding site, which are indicated in bold and
underlined in these figures. The primer binding site of each left
probe of FIGS. 3E and 3H consists of the sequence
GGGTTCCCTAAGGGTTGGA and the primer binding site of each right probe
of FIGS. 3E and 3H consists of the sequence TCTAGATTGGATCTTGCTGGCAC
or TCTAGATTGGATCTTGCTGGCGC. A control nucleotide sequence located
on a nucleic acid molecule according to the invention preferably
has at least 70% sequence identity with the sequence of a probe of
at least one probe set of FIGS. 3E and/or 3H without the primer
binding sites. More preferably said at least one control nucleotide
sequence has at least 80%, more preferably at least 85%, more
preferably at least 90%, most preferably at least 95% sequence
identity with the sequence of a probe of at least one probe set of
FIGS. 3E and/or 3H without the primer binding sites, i.e. without
the bold and underlined sequences. Said at least one control
nucleic acid sequence is preferably selected from the group of
nucleic acid sequences having at least 70% sequence identity to the
probe sequences, without the primer binding sites, of the probe
sets indicated as Control 1 (IL-4), Control 2 (FGF3), Control 3
(BCAS4), Control 4 (LMNA), Control 8 (GALT), Control 9 (SPG4) and
Control 10 (NF2) in FIG. 3H. As demonstrated in Examples 4 and 5
and FIG. 19, these 7 control genes are particularly suitable for
use for a calibrator according to the invention.
[0102] The use of at least two control nucleic acid sequences which
are at least 70% identical to parts of different control genes is
preferred because this allows for a more accurate determination of
the copy number of a gene of interest. Therefore, a nucleic acid
molecule according to the invention preferably comprises at least
two control nucleic acid sequences, more preferably at least three,
more preferably at least four, more preferably at least five, more
preferably at least six, more preferably at least seven control
nucleic acid sequences. Said control nucleic acid sequences are
preferably selected from the group of nucleic acid sequences having
at least 70% sequence identity to the probe sequences, without the
primer binding sites, of the probe sets indicated as Control 1
(IL-4), Control 2 (FGF3), Control 3 (BCAS4), Control 4 (LMNA),
Control 8 (GALT), Control 9 (SPG4) and Control 10 (NF2) in FIG. 3H,
or a combination thereof. In a particularly preferred embodiment, a
nucleic acid molecule or vehicle or plasmid according to the
invention comprises control nucleic acid sequences which have at
least 70% sequence identity to the probes of the probe sets
indicated as Control 1 (IL-4), Control 2 (FGF3), Control 3 (BCAS4),
Control 4 (LMNA), Control 8 (GALT), Control 9 (SPG4) and Control 10
(NF2) in FIG. 3H.
[0103] As explained above, a calibrator according to the invention
is particularly suitable for determining the copy number of a gene
of interest in an individual. Therefore, nucleic acid sequences
located on a calibrator according to the invention are preferably
at least 70% identical to part of a gene of interest which is
subject to copy number variation. The invention thus provides a
nucleic acid molecule comprising at least one nucleic acid sequence
with a length of at least 10 nucleotides which is at least 70%
identical to part of a gene of interest, or a complementary
sequence thereof, and at least one control nucleic acid sequence,
wherein at least 80% of said nucleic acid sequences which are at
least 70% identical to part of a gene of interest comprise a
sequence that is identical to, or complementary to, a gene-specific
nucleotide and/or a pseudo-gene specific nucleotide and/or a
gene-specific sequence and/or a pseudogene-specific sequence and/or
an additional polymorphism within said gene or pseudogene, said
polymorphism preferably comprising an SNP, wherein at least one of
said genes of interest is subject to copy number variation in the
human genome. Said control nucleic acid is preferably at least 70%
identical to, or complementary to, a part of a gene which has a
constant copy number in the human genome.
[0104] Preferably each (pseudo)gene-specific nucleic acid sequence
located on a calibrator according to the invention comprises a
sequence that is identical to, or complementary to, a gene-specific
nucleotide and/or a pseudo-gene specific nucleotide and/or a
gene-specific sequence and/or a pseudogene-specific sequence and/or
an additional polymorphism within said gene or pseudogene. Such
nucleic acid sequence is unique for said (pseudo)gene of interest.
Said nucleic acid sequence located on the calibrator can thus be
used to distinguish said specific gene variant from other genes,
such as other gene variants and/or other genes of a gene cluster.
If a nucleic acid molecule according to the invention comprises
nucleic acid sequences that are specific for each (pseudo)gene
variant of a gene cluster of interest, such nucleic acid molecule
can be used to determine the haplotype, including copy number
variation, of said gene cluster for an individual. Thus, in one
embodiment, a calibrator according to the invention is provided
that comprises nucleic acid sequences which together are at least
70% identical to, or complementary to, parts of each gene variant
of a gene cluster of interest. Such calibrator can be used to
determine in one reaction the presence or absence and copy number
of each gene of said gene cluster, for instance of the KIR or HLA
gene cluster. This means that with the use of such calibrator, the
complete haplotype, including gene copy number, of a gene cluster
in a sample of an individual can be determined. Therefore, in a
preferred embodiment, a nucleic acid molecule, vehicle or plasmid
according to the invention is provided that comprises nucleic acid
sequences which together are at least 70% identical to, or
complementary to, parts of each gene of a gene cluster of interest,
or complementary sequences thereof.
[0105] Because a calibrator according to the invention is
particularly suitable for use in an MLPA method according to the
invention, (pseudo)gene-specific nucleic acid sequences located on
the calibrator preferably have the same number of nucleotides as an
MLPA probe set according to the invention depicted in FIG. 3. Thus,
a nucleic acid sequence located on a calibrator according to the
invention, which is at least 70% identical to part of a gene of
interest or to part of a control gene, preferably has a length of
between 40 and 600 nucleotides. As described herein before,
preferably chemically synthesized probes are used with a length of
between 20 and 110 nucleotides because such probes can be
synthesized easily and cost-effective. In view of the fact that
probe sets according to the invention preferably contain 2 or 3
probes, a nucleic acid sequence located on a calibrator according
to the invention, which is at least 70% identical to part of a gene
of interest or to part of a control gene, preferably has a length
of between 40 and 330 nucleotides. Most preferably, such nucleic
acid sequence has a length of between 90 and 300 nucleotides.
Preferably said nucleic acid sequence does not contain the primer
binding sites of probes according to the invention.
[0106] A calibrator according to the invention is also particularly
useful as an internal quality control when determining the presence
or copy number of a (pseudo)gene of interest in a sample of an
individual. Without the use of such a control, if a sample of an
individual is subjected to an amplification reaction, for instance
as part of an MLPA method, the absence of amplified product of a
gene of interest may indicate that said gene of interest is not
present in said sample. However, it is also possible that the
amplification reaction failed.
[0107] As explained before, if a reference sample containing a
calibrator according to the invention comprising a nucleic acid
sequence specific for the same gene of interest is subjected to the
same MLPA method as a sample of an individual, it serves as a
control for the success of an amplification reaction. If no
amplified product is obtained using said sample of said individual,
but amplified product is obtained using said reference sample, it
can be determined that the amplification was successful. In that
case, it can be concluded that said gene of interest is not present
in said sample of said individual. The presence of amplified
product from said reference sample proves that the amplification
reaction was successful.
[0108] On the other hand, if amplified product is not obtained from
said sample of said individual and also not from said reference
sample, it can be concluded that the amplification reaction failed.
If the amplification reaction succeeded, at least in the reference
sample amplified product should be present.
[0109] Of course, if amplified product following an amplification
reaction is present both when using said sample of said individual
and when using said reference sample, it can be concluded that said
gene of interest is present in said individual.
[0110] Preferably each nucleic acid sequence located on a
calibrator according to the invention is separated from the
upstream or downstream nucleic acid sequence by a spacer sequence
of at least 5 nucleotides. This allows an efficient hybridization
of MLPA probes of a multiple probe set according to the invention
to a calibrator according to the invention and it allows an
efficient amplification reaction. A "spacer sequence" as used
herein is defined as a nucleotide sequence that it not present in
the probes used in an MLPA reaction. More preferably, each (pseudo)
gene-specific or control gene-specific nucleic acid sequence
located on the calibrator is followed by a spacer sequence of at
least 10, more preferably at least 15, more preferably at least 20
nucleotides. Preferably said spacer sequence consists of at most
100 nucleotides, more preferably at most 80 nucleotides to limit
the size of a nucleic acid molecule according to the invention.
This is not necessary, however: said spacer sequences can be larger
than 100 nucleotides. However, in that case a large calibrator will
be generated which can be disadvantageous, for instance because the
larger the calibrator, the more complicated it is to synthesize.
Therefore in a preferred embodiment a nucleic acid molecule or
vehicle or plasmid according to the invention is provided, wherein
each (pseudo)gene-specific nucleic acid sequence or complementary
sequence thereof and/or each control nucleotide sequence or
complementary sequence thereof is followed from 5' to 3' by a
spacer sequence of between 5 and 100 nucleotides, preferably of
between 20 and 80 nucleotides.
[0111] As described above, a calibrator according to the invention
is particularly suitable for determining the copy number of a
(pseudo)gene of interest or for determining a haplotype of a gene
cluster of interest in an individual. Also provided is therefore a
use of a nucleic acid molecule or vehicle or plasmid according to
the invention for determining a copy number of at least one
(pseudo)gene of interest in an individual and a use of a nucleic
acid molecule or vehicle or plasmid according to the invention for
determining a haplotype of a gene cluster of interest of an
individual.
[0112] Also provided is a method for determining a copy number of
at least one (pseudo)gene of interest of an individual
comprising:
[0113] amplifying a sequence with a length of at least 10
nucleotides of said at least one (pseudo)gene of interest using a
sample of said individual and amplifying a sequence with a length
of at least 10 nucleotides of said at least one (pseudo)gene of
interest using a reference sample, said reference sample comprising
a nucleic acid molecule or a vehicle or a plasmid according to the
invention, and
[0114] amplifying a sequence with a length of at least 10
nucleotides of at least one control gene using said sample of said
individual and amplifying a sequence with a length of at least 10
nucleotides of said at least one control gene using said reference
sample;
[0115] determining a level of amplified product of said sequence of
said at least one (pseudo)gene of interest from said sample of said
individual and determining a level of amplified product of said
sequence of said at least one (pseudo)gene of interest from said
reference sample; and
[0116] determining a level of amplified product of said sequence of
said at least one control gene from said sample of said individual
and determining a level of amplified product of said sequence of
said at least one control gene in said reference sample; and
[0117] comparing said levels of amplified products of said
sequences of said at least one (pseudo)gene of interest with each
other and with said levels of amplified products of said sequences
of said at least one control gene, thereby determining the copy
number of said at least one (pseudo)gene of interest. In one
embodiment a method for determining a haplotype of a gene cluster
of an individual is provided which method comprises determining a
copy number of all genes of said gene cluster with a method of the
invention.
[0118] A calibrator according to the invention is further
particularly suitable to determine the presence or absence and the
copy number of a (pseudo)gene of interest in an individual using an
MLPA or MLPA-like method according to the invention, in which at
least one probe set is used which consists of a left probe, a
middle probe and a right probe. The invention therefore also
provides a method for determining a copy number of at least one
nucleic acid of interest in an individual, comprising the steps
of:
a) adding to a sample of said individual and to a reference sample
comprising a nucleic acid molecule or a vehicle or a plasmid
according to the invention at least two different probe sets, each
probe set comprising:
[0119] a first nucleic acid probe, said first probe comprising a
first nucleic acid sequence complementary to a first region of said
nucleic acid of interest and, located 5' thereof, a
non-complementary nucleic acid sequence comprising a first primer
binding site, and
[0120] a second nucleic acid probe, said second probe comprising a
second nucleic acid sequence complementary to a second region of
said nucleic acid of interest and, located 3' thereof, a
non-complementary nucleic acid sequence comprising a second primer
binding site,
[0121] 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 nucleic acid
of interest, and
[0122] wherein, if said third probe is present in said probe set,
said first and said third region of said nucleic acid of interest
are located essentially adjacent to each other and said third and
said second region of said nucleic acid of interest are located
essentially adjacent to each other, and
[0123] wherein, if said third probe is not present in said probe
set, said first and said second region of said nucleic acid of
interest are located essentially adjacent to each other,
[0124] wherein at least one third nucleic acid probe is
complementary to a region of said nucleic acid of interest
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
b) adding to said sample of said individual and to said reference
sample at least one different probe set, each probe set
comprising:
[0125] a first nucleic acid probe, said first probe comprising a
first nucleic acid sequence complementary to a first region of a
control nucleic acid sequence and, located 5' thereof, a
non-complementary nucleic acid sequence comprising a first primer
binding site, and
[0126] at least a second nucleic acid probe, said second probe
comprising a second nucleic acid sequence complementary to a second
region of said control nucleic acid and, located 3' thereof, a
non-complementary nucleic acid sequence comprising a second primer
binding site, and
c) allowing hybridization of said at least two different probe sets
to complementary nucleic acid of said sample of said individual, d)
allowing hybridization of said at least two different probe sets to
complementary nucleic acid of said reference sample, e) subjecting
nucleic acid of said sample of said individual, and nucleic acid of
said reference sample to a ligation reaction, f) subjecting nucleic
acid of said sample of said individual and nucleic acid of said
reference 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 g) determining whether
amplified nucleic acid is present, thereby determining whether said
at least one nucleic acid sequence of interest and/or said control
nucleic acid is present in said sample of said individual, h)
determining a level of amplified product of said at least one
nucleic acid sequence of interest of said sample of said individual
and a level of amplified product of said at least one nucleic acid
sequence of interest of said reference sample; i) determining a
level of amplified product of said at least one control nucleic
acid sequence of said sample of said individual and a level of
amplified product of said at least one control nucleic acid
sequence of said reference sample; j) comparing said levels of
amplified product of said at least one nucleic acid of interest
with said levels of amplified product of said at least one control
nucleic acid, thereby determining the copy number of said at least
one nucleic acid of interest.
[0127] As described above, 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. A
calibrator according to the invention is therefore particularly
suitable for determining the presence and/or copy number of a KIR
gene.
[0128] If the presence or absence of a specific (pseudo)gene is
correlated with (predisposition to) disease, it is often sufficient
to compare a sample of an individual with a reference sample of
which it is known that the specific gene is present. However, the
presence or absence of several KIR genes is not directly correlated
with disease. An individual may lack one or more KIR genes without
this resulting in disease. Importantly, the correlation between one
or more specific KIR genes and disease or the predisposition to
disease often depends on the copy number of the KIR gene. For
instance, a copy number of 1 of a specific KIR gene is not
correlated with a disease, but a copy number of 2 or more of this
KIR gene results in, or predisposes to, disease. As an example, a
higher copy number of KIR2DL2 and/or KIR2DS2 in an individual has
been demonstrated to be predisposing for rheumatoid arthritis with
extra-articular manifestations and rheumatoid vasculitis. Thus,
obtaining information about the presence or absence of a specific
KIR gene in an individual may not be sufficient to obtain
information about the correlation between the KIR gene profile of
an individual and the correlation to disease. It is also necessary
to determine the copy number of KIR genes when information about
such correlations are needed.
[0129] As described in Example 4, the present inventors constructed
a calibrator according to the invention comprising nucleic acid
sequences which are identical to parts of each currently known KIR
gene. The sequence of this calibrator is depicted in FIG. 19. Such
KIR calibrator has been demonstrated to be particularly suitable
for determining the copy number of KIR genes and determining the
complete KIR haplotype of an individual using a method according to
the invention, as is shown in Example 5. Therefore, in a preferred
embodiment, a calibrator according to the invention is specifically
designed for determining the copy number of at least one KIR gene.
Such calibrator is particularly useful in a MLPA method according
to the invention using KIR-specific probe sets. Preferably,
sequences of the probes of the KIR-specific probe sets depicted in
FIG. 3A, 3B, 3C, 3D, 3F or 3G are used. A calibrator according to
the invention that is specifically designed for determining the
copy number of at least one KIR gene therefore preferably comprises
at least one nucleic acid sequence which has at least 70% sequence
identity with a KIR probe set according to the invention or with
sequences complementary thereto. That way, a probe set according to
the invention specific for a particular KIR gene can be used to
hybridize not only to that particular KIR gene in a sample of an
individual, but also to a nucleic acid sequence located on a
calibrator according to the invention corresponding to part of said
particular KIR gene.
[0130] The left and right probes of the probe sets of FIGS. 3A, 3B,
3C, 3D, 3F and 3G contain a primer binding site, which are
indicated in bold and/or underlined in these figures. The primer
binding site of each left probe of FIGS. 3A, 3B, 3C, 3D, 3F and 3G
consists of the sequence GGGTTCCCTAAGGGTTGGA and the primer binding
site of each right probe of FIGS. 3A, 3B, 3C, 3D, 3F and 3G
consists of the sequence TCTAGATTGGATCTTGCTGGCAC or
TCTAGATTGGATCTTGCTGGCGC. A nucleotide sequence located on a nucleic
acid molecule according to the invention preferably has at least
70% sequence identity with a sequence of a probe of at least one
probe set of FIGS. 3A, 3B, 3C, 3D, 3F and/or 3G without the primer
binding sites, i.e. without the bold and/or underlined sequences.
More preferably said at least one nucleotide sequence has at least
80%, more preferably at least 85%, more preferably at least 90%,
most preferably at least 95% sequence identity with a sequence of a
probe of at least one probe set of FIGS. 3A, 3B, 3C, 3D, 3F and/or
3G without the primer binding sites.
[0131] The invention therefore provides a nucleic acid molecule
comprising a nucleotide sequence which has at least 70% sequence
identity with at least one nucleic acid sequence consisting of:
[0132] a) a probe set of FIGS. 3A, 3B, 3C, 3D, 3F and/or 3G,
without the primer binding sites GGGTTCCCTAAGGGTTGGA and
TCTAGATTGGATCTTGCTGGCAC and TCTAGATTGGATCTTGCTGGCGC of said probe
sets, or
[0133] b) a complementary sequence of said probe set without said
primer binding sites,
wherein said nucleic acid sequence of a) or b) either comprises
immediately adjacent to each other:
[0134] the sequences or complementary sequences of a left probe of
said probe set, without primer binding site GGGTTCCCTAAGGGTTGGA,
and of a right probe of the same probe set, without primer binding
site TCTAGATTGGATCTTGCTGGCAC or TCTAGATTGGATCTTGCTGGCGC, if said
probe set consists of two probes, or
[0135] the sequences or complementary sequences of a left probe of
said probe set, without primer binding site GGGTTCCCTAAGGGTTGGA,
and of a middle probe and right probe of the same probe set,
without primer binding site TCTAGATTGGATCTTGCTGGCAC or
TCTAGATTGGATCTTGCTGGCGC, if said probe set consists of three
probes. Such nucleic acid molecule is herein also defined as "a
nucleic acid molecule comprising a nucleic acid sequence of the
probes of at least one probe set of FIGS. 3A, 3B, 3C, 3D, 3F and/or
3G". Preferably, a nucleotide sequence according to the invention
has at least 75%, more preferably at least 80%, more preferably at
least 85%, more preferably at least 90%, most preferably at least
95% sequence identity with said nucleic acid sequence consisting of
a probe set of FIGS. 3A, 3B, 3C, 3D, 3F and/or 3G without the
primer binding sites GGGTTCCCTAAGGGTTGGA and
TCTAGATTGGATCTTGCTGGCAC or TCTAGATTGGATCTTGCTGGCGC, or a
complementary sequence of said probe sets without the primer
binding sites. In one embodiment, a nucleotide sequence according
to the invention consists of the sequence of at least one probe set
of FIGS. 3A, 3B, 3C, 3D, 3F and/or 3G without the primer binding
sites, or a complementary sequence thereof.
[0136] Hence, in a preferred embodiment a calibrator according to
the invention comprises the sequences of all probes of a given
probe set of FIG. 3 adjacent to each other. If, for instance, said
at least one probe set of FIGS. 3A, 3B, 3C, 3D, 3F and/or 3G
consists of a left probe and a right probe, said nucleotide
sequence thus consists from 5' to 3' of: the nucleotide sequence of
said left probe without the primer binding site
GGGTTCCCTAAGGGTTGGA, followed by the nucleotide sequence of said
right probe without the primer binding site TCTAGATTGGATCTTGCTGGCAC
or TCTAGATTGGATCTTGCTGGCGC, or sequences having at least 70%
identity thereto. If said at least one probe set of FIGS. 3A, 3B,
3C, 3D, 3F and/or 3G consists of a left probe, a middle probe and a
right probe, said nucleotide sequence consists from 5' to 3' of:
the nucleotide sequence of said left probe without the primer
binding site GGGTTCCCTAAGGGTTGGA, followed by the nucleotide
sequence of said middle probe, followed by the nucleotide sequence
of said right probe without the primer binding site
TCTAGATTGGATCTTGCTGGCAC or TCTAGATTGGATCTTGCTGGCGC, or sequences
having at least 70%, more preferably at least 75%, more preferably
at least 80%, more preferably at least 85%, more preferably at
least 90%, most preferably at least 95% identity thereto.
[0137] If the copy number of a KIR gene is to be determined, the
level of amplification product of said KIR gene using a sample of
an individual and the level of amplification product of a
corresponding sequence in a reference sample are preferably
compared with an expression level of a control nucleic acid. Also
provided is therefore a nucleic acid molecule comprising a
nucleotide sequence which has at least 70% sequence identity with
at least one nucleic acid sequence consisting of:
[0138] a) a probe set of FIGS. 3A, 3B, 3C, 3D, 3F and/or 3G,
without the primer binding sites GGGTTCCCTAAGGGTTGGA and
TCTAGATTGGATCTTGCTGGCAC and TCTAGATTGGATCTTGCTGGCGC of said probe
sets, or
[0139] b) a complementary sequence of said probe set without said
primer binding sites,
wherein said nucleic acid sequence of a) or b) either comprises
immediately adjacent to each other:
[0140] the sequences or complementary sequences of a left probe of
said probe set, without primer binding site GGGTTCCCTAAGGGTTGGA,
and of a right probe of the same probe set, without primer binding
site TCTAGATTGGATCTTGCTGGCAC or TCTAGATTGGATCTTGCTGGCGC, if said
probe set consists of two probes, or
[0141] the sequences or complementary sequences of a left probe of
said probe set, without primer binding site GGGTTCCCTAAGGGTTGGA,
and of a middle probe and right probe of the same probe set,
without primer binding site TCTAGATTGGATCTTGCTGGCAC or
TCTAGATTGGATCTTGCTGGCGC, if said probe set consists of three
probes, said nucleic acid molecule further comprising at least one
control nucleic acid sequence or a complementary sequence thereof.
Said nucleic acid molecule can be used as such. However, such
nucleic acid molecule can also be present in a vehicle such as a
plasmid, which optionally comprises other nucleic acid sequences.
Also provided is therefore a vehicle or a plasmid comprising a
nucleic acid molecule according to the invention. Such nucleic acid
molecule as such or vehicle or plasmid comprising such nucleic acid
molecule are herein also referred to as "KIR calibrator" according
to the invention.
[0142] Generally, one will be interested in determining the copy
number of more than one KIR gene, for instance for determining the
KIR haplotype of an individual, or for determining predisposition
to a disorder which is associated with the presence or absence or
copy number of more than one KIR gene. A KIR calibrator according
to the invention has the advantage that multiple nucleic acid
sequences, each of which are at least 70% identical to part of a
given KIR gene of interest, are included. Preferably, sequences
specific for all known KIR genes are located on a calibrator
according to the invention. Thus, a KIR calibrator preferably
comprises for each known KIR gene a nucleic acid sequence which is
at least 70% identical to a part with a length of at least 10
nucleotides of said KIR gene. In that case, only one KIR calibrator
according to the invention needs to be present in a reference
sample to determine the copy number of all KIR genes of
interest.
[0143] A KIR calibrator may consist of a single nucleic acid
molecule according to the invention. However, a KIR calibrator may
also comprise multiple nucleic acid molecules according to the
invention. Most preferably, but not necessary, however, a KIR
calibrator according to the invention consist of one vehicle or
plasmid according to the invention. A nucleic acid molecule
according to the invention therefore preferably comprises nucleic
acid sequences of the probes without primer binding sites of at
least two probe sets of FIGS. 3A, 3B, 3C, 3D, 3F and/or 3G, more
preferably nucleic acid sequences of at least three, more
preferably at least four, more preferably at least five, more
preferably at least six, more preferably at least 7, more
preferably at least 8, more preferably at least 9, more preferably
at least 10 probe sets of FIGS. 3A, 3B, 3C, 3D, 3F and/or 3G. In a
particularly preferred embodiment, a nucleic acid molecule
according to the invention comprises nucleic acid sequences of the
probes without primer binding sites GGGTTCCCTAAGGGTTGGA and
TCTAGATTGGATCTTGCTGGCAC or TCTAGATTGGATCTTGCTGGCGC of all probe
sets of FIGS. 3A, 3B, 3C, 3D, 3F and/or 3G, or complementary
sequences thereof. Preferably a nucleic acid molecule according to
the invention comprises nucleic acid sequences that are at least
70% identical to, or complementary to, all probes of FIGS. 3F
and/or 3G, most preferably of FIGS. 3F and 3G, without the primer
binding sites. Provided therefore is a nucleic acid molecule
according to the invention, comprising sequences selected from the
group consisting of:
[0144] all nucleic acid sequences of FIG. 3A without the primer
binding sites GGGTTCCCTAAGGGTTGGA and TCTAGATTGGATCTTGCTGGCAC and
TCTAGATTGGATCTTGCTGGCGC, or sequences having at least 70% sequence
identity to said nucleic acid sequences, and
[0145] all nucleic acid sequences of FIG. 3B without the primer
binding sites GGGTTCCCTAAGGGTTGGA and TCTAGATTGGATCTTGCTGGCAC and
TCTAGATTGGATCTTGCTGGCGC, or sequences having at least 70% sequence
identity to said nucleic acid sequences, and
[0146] all nucleic acid sequences of FIG. 3C without the primer
binding sites GGGTTCCCTAAGGGTTGGA and TCTAGATTGGATCTTGCTGGCAC and
TCTAGATTGGATCTTGCTGGCGC, or sequences having at least 70% sequence
identity to said nucleic acid sequences, and
[0147] all nucleic acid sequences of FIG. 3D without the primer
binding sites GGGTTCCCTAAGGGTTGGA and TCTAGATTGGATCTTGCTGGCAC and
TCTAGATTGGATCTTGCTGGCGC, or sequences having at least 70% sequence
identity to said nucleic acid sequences, and
[0148] all nucleic acid sequences of FIG. 3F without the primer
binding sites GGGTTCCCTAAGGGTTGGA and TCTAGATTGGATCTTGCTGGCAC and
TCTAGATTGGATCTTGCTGGCGC, or sequences having at least 70% sequence
identity to said nucleic acid sequences, and
[0149] all nucleic acid sequences of FIG. 3G without the primer
binding sites GGGTTCCCTAAGGGTTGGA and TCTAGATTGGATCTTGCTGGCAC and
TCTAGATTGGATCTTGCTGGCGC, or sequences having at least 70% sequence
identity to said nucleic acid sequences, and
[0150] any combination thereof, and
[0151] any complementary sequences thereof. Such preferred KIR
calibrator comprises a nucleic acid sequence of part of each KIR
gene currently known. A reference sample comprising such KIR
calibrator is thus particularly suitable to determine the presence
or absence and copy number of each currently known KIR gene and can
thus be used to determine the KIR haplotype, including gene copy
number, of any individual. This is for instance demonstrated in
Example 5, which describes the determination of the KIR haplotype,
including copy number variation, of two siblings of two different
families using a KIR calibrator according to the invention. Without
the use of such KIR calibrator, it would have been only possible to
determine presence or absence of each KIR gene and not the absolute
copy number, because no reference samples are currently available
comprising known quantities of all KIR genes.
[0152] The sequence of the probes, without primer binding sites, of
all probes sets of FIGS. 3F and 3G or sequences complementary
thereto are particularly suitable for use in a KIR calibrator
according to the invention as demonstrated in Examples 4 and 5 and
FIG. 19. Such KIR calibrator has been used by the present inventors
to determine the KIR haplotype and KIR copy number in multiple
families.
[0153] The construction of a non-limiting example of a KIR
calibrator according to the invention is described in Example 4.
This specific KIR calibrator comprises nucleic acid sequences
corresponding to part of each currently known KIR gene, and nucleic
acid sequences corresponding to part of seven control genes which
are known to have a constant copy number in the human genome of 2.
The KIR calibrator described in Example 4 comprises the sequences
of the probes of all probe sets of FIGS. 3F and 3G without the
primer binding sites (i.e. without the bold and underlined part of
the sequences). In this examples, for some probe sequences the
sequences depicted in FIGS. 3F and 3G are used, and for some probe
sequences the sequences that are complementary to the depicted
sequences of FIGS. 3F and 3G are used. As the target nucleic acid
in the test sample is double stranded DNA, both the sense or
antisense sequences can be used. The calibrator depicted in Example
4 further comprises the sequences of the probes of the probe sets
indicated as Control 1 (IL-4), Control 2 (FGF3), Control 3 (BCAS4),
Control 4 (LMNA), Control 8 (GALT), Control 9 (SPG4) and Control 10
(NF2) in FIG. 3H. Again, for some probes the complementary
sequences are used. The sequence of this specific KIR calibrator of
Example 4 is depicted in FIG. 19. This KIR calibrator has been
demonstrated in Example 5 to be particularly suitable for
determining the copy number of KIR genes and determining the
complete KIR haplotype in individuals using MLPA. In one
embodiment, the invention therefore provides a nucleic acid
molecule comprising a sequence which has at least 70% sequence
identity with the sequence depicted in FIG. 19. Preferably said
sequence has at least 75%, more preferably at least 80%, more
preferably at least 85%, more preferably at least 90%, more
preferably at least 95% sequence identity with the sequence
depicted in FIG. 19. In one embodiment, a nucleic acid molecule is
provided which comprises a sequence depicted in FIG. 19.
[0154] A KIR calibrator according to the invention may have spacer
sequences between nucleic acid sequences or complement thereof
which correspond to part of a KIR gene and/or a control gene.
Preferably, in such nucleic acid molecule, most variation in
sequence is allowed in spacer nucleic acid sequences. Nucleic acid
sequences which correspond to part of a KIR gene or to part of a
control gene or complement thereof preferably have at least 70%,
more preferably at least 75%, more preferably at least 80%, more
preferably at least 85%, more preferably at least 90%, more
preferably at least 95% sequence identity with the corresponding
sequence in FIG. 19. The spacer sequences are allowed to have a
sequence which differs more than 70% from the corresponding
sequence in FIG. 19. Because these spacer sequences only serve as
spacers between gene-specific sequences, their nucleotide sequence
has no particular function. No probes need to hybridize to these
spacer sequences during an MLPA reaction. In fact, hybridization to
these spacer sequences is unwanted. Therefore the exact nucleotide
sequence is not important, as long as these spacer sequences are
not complementary to the probes and primers that are used in the
MLPA reaction. Thus, the sequence identity between a spacer
sequence in a KIR calibrator according to the invention and the KIR
calibrator of FIG. 19 can be less than 60%, less than 50%, less
than 40%, less than 30%, less than 25% or even lower.
[0155] The invention also provides a use of a nucleic acid molecule
or a vehicle or plasmid according to the invention for determining
the copy number of at least one KIR gene in an individual and/or
for determining a KIR haplotype of an individual.
[0156] The invention further provides method for determining the
copy number of at least one KIR gene of an individual
comprising:
[0157] amplifying a sequence with a length of at least 10
nucleotides of said at least one KIR gene using a sample of said
individual and amplifying a sequence with a length of at least 10
nucleotides of said at least one KIR gene using a reference sample,
said reference sample comprising a nucleic acid molecule or a
plasmid according to the invention, and
[0158] amplifying a sequence with a length of at least 10
nucleotides of at least one control gene using said sample of said
individual and amplifying a sequence with a length of at least 10
nucleotides of said at least one control gene using said reference
sample;
[0159] determining a level of amplified product of said sequence of
said at least one KIR gene from said sample of said individual and
determining a level of amplified product of said sequence of said
at least one KIR gene from said reference sample; and
[0160] determining a level of amplified product of said sequence of
said at least one control gene from said sample of said individual
and determining a level of amplified product of said sequence of
said at least one control gene in said reference sample; and
[0161] comparing said levels of amplified products of said
sequences of said at least one KIR gene with each other and with
said levels of amplified products of said sequences of said at
least one control gene, thereby determining the copy number of said
at least one KIR gene.
[0162] Said part of said at least one KIR gene and said part of
said at least one control gene preferably comprise at least 10,
more preferably at least 15, more preferably at least 18, more
preferably at least 19, more preferably at least 20 nucleotides. As
described herein before, in an MLPA method according to the
invention, preferably chemically synthesized MLPA probes are used
with a length of between 20 and 110 nucleotides because such probes
can be synthesized easily and cost-effective. Therefore, if the
copy number of at least on KIR gene is determined using MLPA, the
KIR gene-specific and control gene-specific sequences located on a
calibrator according to the invention preferably have a length of
between 40 and 330 nucleotides. Most preferably, such nucleic acid
sequences have a length of between 90 and 300 nucleotides.
[0163] As described herein before, a KIR calibrator is particularly
suitable for determining the KIR haplotype and/or copy number of
KIR genes using an MLPA method. Therefore, in a preferred
embodiment a method according to the invention further comprises
the steps of:
a) adding to said sample of said individual and to said reference
sample at least one probe set selected from FIGS. 3A, 3B, 3C, 3D,
3F and/or 3G, and b) optionally, adding to said sample of said
individual and to said reference sample at least one probe set
selected from FIG. 3E or 3H, and c) allowing hybridization of said
probe set or probe sets to complementary nucleic acid of said
sample of said individual, and d) allowing hybridization of said
probe set or probe sets to complementary nucleic acid of said
reference sample, and e) subjecting nucleic acid of said sample of
said individual, and nucleic acid of said reference sample to a
ligation reaction.
[0164] Said method preferably further comprises amplifying ligated
nucleic acid and determining levels of amplified products, thereby
determining the copy number of at least one KIR gene of said
individual. In a preferred embodiment, a method according to the
invention comprises the use of at least one of the probe sets
selected from FIGS. 3A, 3B, 3C, 3D, 3F and/or 3G that comprise a
third nucleic acid probe.
[0165] The invention also provides a method for determining a KIR
haplotype of an individual comprising determining the copy number
of at least 5, preferably at least 10, more preferably at least 15,
most preferably all KIR genes of said individual with a method
according to the invention.
[0166] It is described herein before in detail how the copy number
of a gene is determined based on the level of amplified product of
part of said gene in a test sample and in a reference sample and
the level of amplified product of part of at least one control gene
in said test sample and said reference sample. Briefly, the
difference between the peak intensities of each amplified control
gene product is determined by comparing for each control gene the
intensity peak of amplified product in a test sample and the
intensity peak of amplified product in a reference sample. The
difference between the peak intensity of each amplified KIR gene is
also determined by comparing for each KIR gene the intensity peak
of amplified product in said test sample and the intensity peak of
amplified product in said reference sample. Subsequently, the copy
number of the KIR gene is determined based on the proportions of
the peak intensities of the KIR gene in test and reference sample
and the proportion of the peak intensities of the control gene in
test and reference sample.
[0167] 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 FIGS. 3A, 3B, 3C, 3D, 3F and/or 3G 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 FIGS. 3C, 3D, 3F and/or 3G are used to detect KIR2DS3
and/or KIR2DS4 polymorphisms. With probes selected from FIG. 3
predisposition to leukemia subtypes is thus particularly well
determined.
[0168] 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 FIGS. 3A, 3B, 3C, 3D, 3F and/or 3G, 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 FIGS. 3C, 3D, 3F and/or 3G 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.
[0169] 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.
[0170] 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
FIGS. 3A, 3B, 3C, 3D, 3F and/or 3G 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 FIGS. 3C, 3D, 3F and/or 3G 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.
[0171] 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 FIGS. 3A, 3B, 3C, 3D, 3F
and/or 3G, 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 FIGS. 3C, 3D, 3F and/or 3G are used
for determining the presence or absence of KIR polymorphisms.
[0172] 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 HW, 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.
[0173] 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 FIGS. 3A, 3B, 3C, 3D, 3F and/or 3G
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 FIGS. 3C, 3D, 3F and/or 3G are used
to estimate the copy number of KIR3DL1 and/or KIR3DS1 and/or KIR
2DL3. 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.
[0174] 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 or 3F or 3G,
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 FIGS. 3C, 3D, 3F and/or 3G are used
for determining the copy number of KIR genes.
[0175] 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 FIGS. 3A, 3B, 3C, 3D, 3F and/or 3G 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
FIGS. 3C, 3D, 3F and/or 3G 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
[0176] 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 or 3F or 3G, 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 FIGS. 3C, 3D, 3F and/or 3G are used for determining the
presence or absence of KIR polymorphisms.
[0177] 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).
[0178] 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 FIGS. 3A, 3B, 3C, 3D, 3F and/or 3G 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 FIGS. 3C, 3D, 3F and/or 3G 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 FIGS. 3C, 3D, 3F and/or
3G 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.
[0179] 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 FIGS. 3A, 3B, 3C, 3D, 3F and/or 3G, 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 FIGS. 3C, 3D, 3F and/or 3G are used for determining the
copy number of KIR genes.
[0180] 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.
[0181] 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 or 3F or 3G,
and b) correlating the result obtained in step a) with presence or
absence of said predisposition.
[0182] 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 FIGS. 3A, 3B,
3C, 3D, 3F and/or 3G, 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 FIGS. 3C, 3D, 3F and/or 3G are used for determining the
copy number of KIR genes.
[0183] 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
[0184] FIG. 1. A) Schematic outline of a conventional MLPA
reaction. The figure is adapted from www.mpla.com. 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.
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.
[0185] FIG. 2. IUB nucleotide codes of degenerate bases
[0186] FIG. 3 KIR-specific probe sets. A) KIR probe mix 1. Bold and
underlined nucleotides represent primer binding sites., B) KIR
probe mix 2. Bold and underlined nucleotides represent primer
binding sites., C) extended KIR probe mix 1. Bold and underlined
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 and underlined 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. Bold and underlined
nucleotides represent primer binding sites, F) improved KIR probe
mix 1. Bold and underlined nucleotides represent primer binding
sites, G) improved KIR probe mix 2. Bold and underlined nucleotides
represent primer binding sites, H) improved control probe mix. Bold
and underlined nucleotides represent primer binding sites.
[0187] 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.
[0188] 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).
[0189] 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).
[0190] 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.
[0191] 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).
[0192] FIG. 9. Comparison of peak intensities of the probe 2DS2
(black arrows) between a true positive for KIR2DS2 (top) and a
false positive (bottom).
[0193] 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.
[0194] FIG. 11. The pedigree of family 1347.
[0195] 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.
[0196] 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).
[0197] 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).
[0198] 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).
[0199] FIG. 12. The pedigree of family 1349.
[0200] 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.
[0201] 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).
[0202] 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).
[0203] 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).
[0204] FIG. 13. Detection of KIR alleles and KIR copy number
variation.
[0205] FIG. 14. Schematic representation of the process to design
synthetic genes for the KIR MLPA calibrator. The process includes
design of the sequence, design of the oligo's, assembly of the
oligo's by means of ligation and amplification of the assembled
gene.
[0206] FIG. 15. Agarose gels showing the PCR products after oligo
assembly (step 1) and gene amplification (step 2). Left: The
assembly and amplification of the entire 1.1 kb gene failed. Right:
The gene was split into two smaller genes, A and B, with 180 bp
overlap. These products were amplified successfully and
subsequently combined to form one gene of 1,1 kb.
[0207] FIG. 16. pBlueScript is digested so that newly synthesized
genes can be combined into one plasmid, resulting in the KIR MLPA
calibrator.
[0208] FIG. 17. Probes designed to recognize specific KIR genes,
based on variation of one or two specific base pairs. The probe
binding sites are arranged to prevent interference: either with a
spacer (nonsense sequence) between the binding sites, or by
alternating between the binding site between the sense and
anti-sense strands.
[0209] FIG. 18. KIR haplotype of CEPH families 3144 (A) and 3149
(B).
[0210] FIG. 19. Sequence of KIR MLPA calibrator. Small letters
represent the backbone of the pBlueScript plasmid and capitals
represent nucleotide sequences which are inserted into the plasmid
as described in Example 4, including KIR (pseudo)gene specific
sequences, control gene specific sequences, and spacer
sequences.
EXAMPLES
Example 1
[0211] This Example presents a new method for KIR genotyping.
[0212] 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.
[0213] 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.
[0214] 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.
[0215] 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.
[0216] Some of these activating KIRs seem to have lower affinity
for their cognate HLA class I ligands in comparison with their
related inhibitory receptors.
[0217] 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
[0218] 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 extra cellular
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 cytoplamic 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).
[0219] 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
[0220] 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.
[0221] 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
[0222] 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).
[0223] 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
[0224] 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.
[0225] 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
[0226] 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.
[0227] 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 3DL 1*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
[0228] 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.
[0229] 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.
[0230] 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.
[0231] 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
[0232] 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 .quadrature. including 2 parents and 2
children (table 4: KIR typing of the 48 samples and FIG. 7: the
pedigrees) .quadrature. 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
[0233] 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'.
[0234] 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.
[0235] 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.
[0236] 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
[0237] All DNA samples were diluted to 20 ng/.mu.l with water and 5
.mu.lwas denatured at 98.degree. C. for 5 minutes in 200 .mu.l
tubes in a Biometra T-1 Thermoblock with heated lid. 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
[0238] 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
[0239] 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
[0240] 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.
[0241] 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. 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.
[0242] 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 bp 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).
[0243] 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.
[0244] 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.
[0245] 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
[0246] 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.
[0247] 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
[0248] 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.
[0249] 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
[0250] 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
[0251] 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.
[0252] 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
[0253] 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)
[0254] 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
[0255] 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
[0256] 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
[0257] 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
[0258] 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 by
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
[0259] 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
[0260] 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).
[0261] 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).
[0262] 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
[0263] 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
[0264] 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
[0265] 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
[0266] 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.
[0267] 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.
[0268] 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.
[0269] 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 TUB codes
in the probe sequence will overcome the problem of
misinterpretation of copy number differences between
individuals.
[0270] 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
[0271] Before the present invention, the main problem in designing
synthetic MLPA probes for KM 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.
[0272] 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.
[0273] 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.
[0274] 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
[0275] 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
[0276] 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
[0277] With the extended probesets 1 and 2 all KIR genes and
several KIR gene variants were detected.
[0278] 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.
[0279] 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
[0280] 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
[0281] 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)
[0282] 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
[0283] 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
[0284] From the MLPA data within pedigrees haplotyping can be
inferred. First of all, the framework genes KIR3DL3 and KIR3DPI 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.
[0285] In family 1347, we have deduced, using the extended
probesets, from the pedigree a correct and complete KIR haplotype
analysis (FIG. 11B).
[0286] 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 .quadrature. for instance
.quadrature. 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).
[0287] 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).
[0288] 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.
[0289] 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.
[0290] SSP-PCR can not discriminate between 3DL1 variants (also not
between 3DS1 variant genes nor 2DL4 variant genes).
[0291] 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". 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.
[0292] 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).
[0293] 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.
[0294] 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.
[0295] 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).
[0296] 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.
[0297] For the SSP PCR data the two haplotype models are shown to
interpret possible CNV results, resp. the conventional KIR
haplotype model in FIGS. 11B1 and 12B1 and the novel KIR haplotype
model in FIGS. 11B2 and 12B2. FIGS. 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.
[0298] 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.
[0299] 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.
[0300] 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.
[0301] 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.
[0302] 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).
[0303] 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
[0304] 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
[0305] 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.
[0306] 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.
[0307] Materials & Methods
[0308] 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-
GGGTTCCCTAAGGGTTGGACAAGGACCCCTCACGCCTCGTTGGAC-3' 711B - KIR3DL1
Middle probe part: 5'-PO4-
GATCCATGATGGGGTCTCCAAGGCCAATTTCTCCATCGGTCCCATGATG CT-3' 711C -
KIR3DL1 Right probe part: 5'-PO4-
GCCCTTGCAGGGACCTACAGATGCTACGGTTCTGGTCTAGATTGGATCT TGCTGGCAC-3'
[0309] For DNA selection/isolation, probe design, MLPA reaction,
electrophoresis and analysis see materials & methods of example
1.
[0310] 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
[0311] 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.
[0312] 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.
[0313] 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.
Example 4
Introduction
[0314] To be able to determine copy number variation of the KIR
gene family using MLPA, it is necessary to compare the data with a
sample of which it is exactly known how many copies of each KIR
gene is present. Up till now, there was no such sample available.
That is why a calibrator for the KIR MLPA was designed.
[0315] The calibrator is a DNA construct that is designed to
contain one binding site for each probe set in the KIR MLPA. If
this construct is run in an MLPA just as any other sample, it will
result in a peak pattern which resembles the presence of one copy
per KIR gene. In theory, if the concentration of this construct is
prepared twice as high as compared to the (estimated) concentration
of a human genome, the amount of copies that one peak represents is
equal to two. The peak pattern of each human genome can be compared
to the peak pattern of the calibrator and so the amount of copies
of each KIR per genome can be determined.
Materials & Methods
[0316] The calibrator is constructed as follows: A total of six
genes (FIG. 16) containing the binding sites of all probes were
synthesized. These genes were synthesized using a modified version
of the method described in an article by Stemmer et al. (Gene,
1995) as schematically shown in FIG. 14. The intended sequences of
both the sense and anti-sense strands of each gene were designed
first. All genes included unique digestion sites at both ends for
cloning purposes. These sequences were broken into pieces of
oligo's 60 base pairs long with each 20 base pairs of "non-coding
material" in between. The pieces of the anti-sense strand were
created so that they would have exactly 20 bp overlap with two
pieces of the sense strand.
[0317] The 60-mers were obtained from (Life technologies) and
assembled with the use of a PCR machine. The overlapping part of
the oligo's ligate with each other and the open spaces of 20 bp are
filled up with nucleotides. Part of the product is used for a
second PCR reaction containing only the outside oligo's which
function as primers in this reaction. The assembled gene is
amplified.
Technical Aspects
[0318] A difference between the construction of the calibrator and
the protocol of Stemmer et al. is the length of the starting
oligo's. We have decided on the use of 60-mers, while Stemmer et
al. uses 40-mers. Also, Stemmer et al. predicts that products can
be synthesized of 3-5 kilo bases, while the largest part that we
were able to create with this method is 680 bp. For this
calibrator, six genes were designed of which gene 1 had a length of
892 bp, gene 2 to 5 had lengths of around 1.1 kb and gene 6 had a
length of 2.8 kb. None of these genes could be synthesized in two
steps. Therefore it was required to split the genes into smaller
parts of maximally 680 bp with a 180 bp overlap. The oligo's that
form these smaller parts were assembled together and the products
were amplified. Successfully synthesized parts were combined in a
second amplification reaction to create the entire gene of 1.1 kb.
This process is illustrated by the agarose gel separation of the
PCR products of one of these genes in FIG. 15.
[0319] Genes 1 to 5 were all synthesized by these 3 steps. Gene 6
is larger and requires multiple steps to assemble. This gene was
divided into 6 smaller genes of .about.650 bp which were combined
to form 5 genes of .about.1 kb. These 1 kb products were then
combined to form 4 products of .about.1.5 kb and so forth. The
longer the products, the longer the overlap had to be for the
separate products to ligate to each other. Also, for several steps
it was required to purify the specific product from gel to remove
all side products that had formed.
[0320] When a synthetic gene was produced it was cloned into a
plasmid; either eGFP-C1 or pBlue Script. Sequencing of the inserts
confirmed the presence of the entire gene. Mutations (introduced by
possibly incorrectly formed oligo's and multiple PCR rounds) were
present at a relatively low rate (approx. 2 mutations per kb). When
a mutation was present in the vicinity of a ligation point, a
site-directed mutation kit was used to mutate this specific base
pair back to the original sequence. Only then were all genes
combined into one plasmid: pBlueScript. The plasmid was digested so
that these synthetic genes could be inserted. The result was one
large construct of 11.5 kb as schematically depicted in FIG. 16.
The sequence of the complete KIR MLPA calibrator is depicted in
FIG. 19.
Mode of Action
[0321] During an MLPA reaction the KIR probes will find only one
site on the calibrator to bind. To prevent any interference or
competition between different probe sets, the calibrator was
designed in such a way that there is the least possible overlap
between the probe binding sites. To accomplish this, spacers
(non-coding sequences) were introduced between the probe binding
sites. This method was applied for gene 1 to 5. A second technique
is to alternate the probe binding site between the sense and
anti-sense strand. A schematic representation of these two ways of
distribution is shown in FIG. 17.
[0322] The calibrator contains the probe binding sites of all KIR
probe sets of FIGS. 3F and 3G and control probe sets of FIG. 3H.
This last aspect facilitates an extra quality control for each MLPA
run. If necessary, more probe binding sites can be added to the
construct as several single cutting digestion sites are included in
the construct.
Example 5
Introduction
[0323] The genotyping and CNV detection within CEPH families
enabled us to find further proof of principle and application of
the calibrator-related assessment of the number of KIR genes within
any given individual. As exemplified by two CEPH families 1344 and
1349, the CNV for 3DP1 and 2DL3 has been determined by the current
KIR MLPA probe mixes and corresponding calibrator.
[0324] Previous SSP-PCR and MLPA data gave the same results in the
presence or absence of KIR genes. By the added value of CNV
detection within families, it has become possible to construct and
trace the haplotypes of father and mother which are inherited by
their offspring, as indicated in FIGS. 18A and 18B
[0325] Haplotyping is a means to further validate the MLPA for KIR
genes. Each haplotype (i.e. a series of adjacent genes at one
locus) on one chromosome of the parent is divided among the male
and female germline cells and after conception of the fertilized
egg a set of two haplotypes is created again, i.e. one from the
father and one from the mother.
[0326] The framework genes are present in both the relatively fixed
haplotype A and the highly variable haplotype B (see FIGS. 11B and
12B), which means that these genes are always present in each
individual as a pair of genes. The presence of the other KIR genes
are more variable and can be completely absent or present as a
single or two copies of that gene, depending on the haplotype
(haplotype A or B).
Family Tree Assembly
[0327] A set of previously genotyped families was used to validate
the use of the calibrator of Example 4, the sequence of which is
shown in FIG. 19, in the KIR MLPA. These families, called Centre
d'Etude Polymorphisme Humain (CEPH) families, were typed during the
13.sup.th Immuno Histocompatibility Workshop (IHS) using SSP-PCR.
In this example it is described how the calibrator is applied
during copy number determination in two of these CEPH families.
Materials and Methods
[0328] The KIR MLPA was based upon the method as described in
Example 1. In short, the MLPA was started with a probe
hybridization step, followed by ligation of the different probes.
The final step was the amplification of bound probes with a
polymerase chain reaction (PCR). The PCR was optimized for this
specific MLPA; 10 .mu.l of ligation mixture was added to 40 .mu.l
of PCR mixture. This mixture consisted of 5 .mu.l Accuprime Taq
Buffer, 0.4 .mu.l Accuprime Taq enzymes (Invitrogen, AccuPrime Taq
DNA Polymerase High Fidelity kit), 1.875 mM MgSO4 and 1.5 .mu.l of
Salsa PCR primers (MRC Holland). The PCR reaction created products
that contained a fluorescent label allowing for fragment analysis
by electrophoresis. Fragment analysis was performed with a mixture
of HiDi Formamide (9 .mu.l) and Promega Internal Lane Standard
60-600 (1 .mu.l). The probes were divided among three mixes to
prevent competition between homologous primer sets. The control
probes used in this assay were obtained from MRC Holland.
[0329] The PCR fragments that were formed during the KIR MLPA were
analysed on a sequencer platform, in our case an 3130x1 Genetic
Analyzer from Applied Biosystems. The raw data set was further
analyzed using Genemarker software (Softgenetics LLC). This
software normalized the peak pattern so that each peak gave a
representative height and area for the actual amount of copies of
each specific gene. This was also done for the peak pattern of the
calibrator: the software assumed that two copies of each gene were
present in every sample. Therefore, it adjusted the peaks of the
calibrator (which actually represent one copy of each gene) to
represent two copies.
[0330] The next step of the analysis was the MLPA analysis itself.
The peak pattern of one or more calibrator samples was used as a
reference for two copies. The software compared each peak from a
tested sample with the height or area of the comparable peak in
this reference, resulting in a ratio. A ratio of 0.75-1.25 meant an
equal amount of copies, so 2. A ratio of 0.25-0.75 was marked as a
deletion, so 1 copy. A ratio of 1.25-1.75 referred to a
duplication, so 3 copies.
[0331] For each member of two CEPH families, the copy numbers of
the KIR genes were determined as described above. The genes for the
null-variants for KIR2DL4 and KIR2DS4 were determined, but KIR2DL5A
and KIR2DL5B were not distinguished from each other. As inheritance
of these genes is thought to occur in a Mendelian manner on two
alleles, each copy in the offspring should have its origin in one
of the parents. This allowed us to divide the found copy numbers of
the KIR genes in separate alleles which are inherited from the
parents.
Results and Discussion
[0332] The KIR haplotype of the two families as determined are
shown in FIGS. 18A (family 1344) and 18B (family 1349). The parents
are indicated with -01 and -02, the square represents the father
and the circle represents the mother, with the determined KIR
haplotype besides these figures. The KIR haplotype shows which KIR
genes are present in the parent and the presence and copy number of
the KIR genes on each chromosome is shown, one chromosome left of
the vertical line and the other chromosome to the right of the
vertical line. Below the KIR haplotypes of the parents, those of
two siblings are depicted. The KIR haplotypes indicate which
chromosome is originating from the father (P) and which chromosome
is originating from the mother (M).
[0333] Before the use of the KIR calibrator, only the presence or
absence of a specific KIR gene could be demonstrated. The use of
the KIR calibrator enables determination of the copy number of the
KIR genes as well. For instance, FIG. 18A shows that both parents
of family 1344 carry the KIR2DP1 gene and that both siblings
inherited the gene. However, previously it was unknown how many
copies of this gene were present. It is now demonstrated that both
parents and both siblings in this family carry three copies of the
KIR2DP1 gene in their genome. Apart from 2DP1, similar results for
CNV of 2DL3 is demonstrated in family 1349.
[0334] We further show that the calibrator-based KIR MLPA analysis
can be used to not only indicate CNV of these KIR genes (showing
0-1-2-or-more gene copies) but also to help determine the mode of
inheritance to siblings within one family. This is illustrated for
KIR2DL3 in family 1349 (FIG. 18B). The two siblings tested in this
family differ in the number of KIR2DL3 genes inherited from each of
the parents who both carry 3 copies in their genome.
[0335] Thus, CNV, which can now be determined with a KIR
calibrator, can be a helpful way to determine a certain haplotype
in a pedigree and use it as a tracer for genetic inheritance
patterns within KIR-genotyped families.
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 cl-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 Activating KIRs
Ligands KIRs Ligands 2DL1 HLA-C group 2, 2DS1 HLA-C group 2
allotypes Cw1, allotypes Cw1, 4, 5, 6, 17, 18 4, 5, 6, 17, 18 2DL2
and HLA-C group 1, 2DS2 HLA group 1, 2DL3 allotypes Cw1, allotypes
Cw1, 3, 7, 8, 13, 14 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## 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 Size Concentration Code Probe [bp] Probe
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 Size Concentration Code Probe [bp] Probe
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 Size Concentration Code Probe (Gene)
[bp] Probe 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.
length Concentration code gene [bp] probe part (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 availableon
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 1 Probe set 2 Probe set 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 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
20L4*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
2DL58*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*0005
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*0004 3DL2*013 3DL2*005 3DL2*0017 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*0025 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 ##STR00003## 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 ##STR00004##
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 ##STR00005## 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 ##STR00006##
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
REFERENCES
[0336] Brown M A. Genetics and the pathogenesis of ankylosing
spondylitis. Curr Opin Rheumatol. 2009; 21:318-23. [0337]
Carrington M, Noramn P. The KIR gene cluster 2003. [0338] Chan A T,
Kollnberger S D, Wedderburn L R, Bowness P. Expansion and enhanced
survival of natural killer cells expressing the killer
immunoglobulin-like receptor KIR3DL2 in spondylarthritis. Arthritis
Rheum. 2005;52:3586-95. [0339] Cook M A, Norman P J, Curran M D,
Maxwell L D, Briggs D C, Middleton D, Vaughan R W. A
multi-laboratory characterization of the KIR genotypes of the
10.sup.th International Histocompatibility Workshop cell lines.
Human Immunology 2003: 64, 567-571 [0340] Crum K A, Logue S. E,
Curran M D, Middleton D. Development of a PCR-SSOP approach capable
of defining the natural killer cell inhibitory receptor (KIR) gene
sequence repertoire. Tissue Antigens 2000: 56: 313-326. [0341] Du
Z, Gjertson D W, Reed E F, Rajalingam R. Receptor-ligand analyses
define minimal killer cell Ig-like receptor (KIR) in humans.
Immunogenetics 2007:59:1-15 [0342] Gomez-Lozano N, Gardiner C M,
Parham P, Vilches C. Some human KIR haplotypes contain two KIR2DL5
genes: KIR2DL5A and KIR2DL5B. Immunogenetics 2002: 54 (5): 314-9
[0343] Gomez-Lozano N, Estefania E, Williams F, Halfpenny I,
Middleton D, Solis R, Vilches C. The silent KIR3DP1 gene (CD158c)
is transcribed and might encode a secreted receptor in a minority
of humans, in whom the KIR3DP1, KIR2DL4 and KIR3DL1/KIR3DS1 genes
are duplicated. European Journal Immunology 2005: 35(1):16-24
[0344] Hollenbach J A, Ladner M B, Saeteurn K, Taylor K D, Mei L,
Haritunians T, McGovern D P B, Erlich H A, Rotter J I, Trachtenberg
E A. Susceptibility to Crohn's disease is mediated by
KIR2DL2/KIR2DL3 heterozygosity and the HLA-C ligand. Immunogenetics
2009: 61(10): 663-671 [0345] Hsu K C, Liu X R, Selvakumar A,
Mickelson E, O'Reilly R J, Dupont B. Killer Ig-like receptor
haplotype analysis by gene content: evidence for genomic diversity
with a minimum of six basic framework haplotypes, each with
multiple subsets. Journal of Immunology 2002: 1; 169(9):5118-29
[0346] Hsu K C, Chida S, Geraghty D E, Dupont B. The killer cell
immunoglobulin-like receptor (KIR) genomic region: gene-order,
haplotypes and allelic polymorphism. Immunol Rev. 2002 December;
190: 40-52. [0347] Khakoo S I, Thio C L, Martin M P, Brooks C R,
Gao X, Astemborski J, et al. HLA and NK cell inhibitory receptor
genes in resolving hepatitis C virus infection. Science 2004; 305:
872-4. [0348] Li H, Pascal V, Martin M P, Carrington M, Anderson S
K. Genetic control of variegated KIR gene expression: polymorphisms
of the bi-directional KIR3DL1 promoter are associated with distinct
frequencies of gene expression. PLoS Genet. 2008 November;
4(11):e1000254. [0349] Majorczyk E, Pawlik A, .quadrature.uszczek
W, Nowak I, Wi.quadrature.niewski A, Jasek M,
Ku.quadrature.nierczyk P. Associations of killer cell
immunoglobulin-like receptor genes with complications of rheumatoid
arthritis. Genes Immun. 2007; 8:678-83. [0350] Marsh S, Parham P,
Dupont B, Geraghty D, Trowsdale J, Middelton D, Vilches C,
Carrington M, Witt C, Guethlein L, Shilling H, Garcia C, Hsu K,
Wain H. Killer-cell Immunoglobulin-like Receptor (KIR) Nomenclature
Report. Human Immunology 2002: 64, 648-654. [0351] Martin M P, Qi
Y, Gao X, Yamada E, Martin J N, Pereyra F, et al. Innate
partnership of HLA-B and KIR3DL1 subtypes against HIV-1. Nat Genet
2007; 39:733-40. [0352] Middleton D, Williams F, Halfpenny I A. KIR
genes. Transplant Immunology 2005: 14(3-4):135-42 [0353] Parham P,
McQueen K L. Alloreactive killer cells: hindrance and help for
haematopoietic transplants. Nature reviews Immunology 3 2003: doi:
10.1038/nri999 [0354] Shilling H G, Guethlein L A, Cheng N W,
Gardiner C M, Rodriguez R, Tyan D, Parham P. Allelic polymorphism
synergizes with variable gene content to individualize human KIR
genotype. Journal of Immunology 2002: 1:168(5):2307-15 [0355]
Schouten J P, McElgunn C J, Waaijer R, Zwijnenburg D, Diepvens F,
Pals G. Relative quantification of 40 nucleic acid sequences by
multiplex ligation-dependent probe amplification. Nucleic Acid
Research. 2002: 15:30(12):e57 [0356] Stemmer W P, Crameri A, Ha K
D, Brennan T M, Heyneker H L. Single-step assembly of a gene and
entire plasmid from large numbers of oligodeoxyribonucleotides.
1995 Gene, 164: 49-53. [0357] Sun J Y, Gaidulis L, Miller M M, Goto
R M, Rodriguez R, Forman S J, Senitzer D. Development of a
multiplex PCR-SSP method for Killer-cell immunoglobulin-like
receptor genotyping. Tissue Antigens 2004: 64: 462-468. [0358]
Trowsdale J, Barten R, Haude A, Stewart C A, Beck S, Wilson M J.
The genomic context of natural killer receptor extended gene
families. 2001. Immunological Reviews volume 181: 20-38 [0359]
Urhberg M, Valiante N M, Shum B P, Shilling H G, Lienert-Weidenbach
K, Corliss B, Tyan D, Lanier L L, Parham P. Immunity volume 1997:
7, 753-763 [0360] Uhrberg M. The KIR gene family: life in the fast
lane of evolution. European Journal of Immunology 2005: 35:10-15
[0361] Vilches C, Parham P. KIR: diverse, rapidly evolving
receptors of innate and adaptive immunity. Annual Reviews
Immunology 2002: 20:217-51 [0362] Vilches C, Castano J,
Gomez-Lozano N, Estefania E. facilitation of KIR genotyping by a
PCR-SSP method that amplifies short DNA fragments. 2007. Tissue
Antigens 70, 415-422. [0363] Williams F, Maxwell L D, Halfpenny I
A, Meenagh A, Sleator C, Curran M D, Middleton D. Multiple copies
of KIR 3DL/S1 and KIR 2DL4 genes identified in a number of
individuals. Human Immunology 2003: 64, 729-732. [0364] Yen J H,
Moore B E, Nakajima T, Scholl D, Schaid D J, Weyand C M, Goronzy J
J. Major histocompatibility complex class I-recognizing receptors
are disease risk genes in rheumatoid arthritis. J Exp Med. 2001;
193:1159-67. [0365] Zhang Y, Wang B, Shihui Y, Liu S, Liu M, Shen
C, Teng Y, Qi J. Killer cell immunoglobulin-like receptor gene
polymorphisms in patients with leukemia: Possible association with
susceptibility to the disease. Leuk Res 2009, doi10.1016/j
leukres.2009.04.022.
Sequence CWU 1
1
43119DNAArtificialprimer 1gggttcccta agggttgga
19223DNAArtificialprimer 2tctagattgg atcttgctgg cac
23318DNAArtificialprimer 3gggttcccta agggttgg
18448DNAArtificialprobe 4gggttcccta agggttggac tgaccttggg
ccctgcagag aacctaca 48548DNAArtificialprobe 5ttcatgggcc tccccctccc
tggatgtcta gattgatctt gctggcac 48650DNAArtificialprobe 6gggttcccta
agggttggat agatgcttcg gctctttccg tgccctgccc 50750DNAArtificialprobe
7cacgcgtggt cagacccgag tgacccgtct agattggatc ttgctggcac
50854DNAArtificialprobe 8gggttcccta agggttggac catcacgatg
tccagagggt cactgggagc tgaa 54954DNAArtificialprobe 9aactgatagg
gggagtgagg aacagagacc gtctagattg gatcttgctg gcac
541055DNAArtificialprobe 10gggttcccta agggttggaa tccaccctaa
ggtttgggga aggactcacc catga 551156DNAArtificialprobe 11gtggccaggc
cccctgcagc aagaagaacc ctgtctagat tggatcttgc tggcgc
561265DNAArtificialprobe 12gggttcccta agggttggac ccaaggtggt
caggacaagc ccttgctgtc tgcctggccc 60agctc 651356DNAArtificialprobe
13tgtggtgcct ccaggacatg tgattcttcg gtgtctagat tggatcttgc tggcgc
561466DNAArtificialprobe 14gggttcccta agggttggac accatgatca
ccagggggtt gctgggtgct gaccacccag 60tgagga 661559DNAArtificialprobe
15agtgtgggtg tgaaccccga catctgtagg tccctgtcta gattggatct tgctggcgc
591661DNAArtificialprobe 16gggttcccta agggttggac tcccctctct
gtgcagaagg aagtgctcaa acatgacatc 60c 611776DNAArtificialprobe
17gaccaacatt gcaggatgac tgtctcttct gatttcacca ggtgacctgg gagtctagat
60tggatcttgc tggcac 761857DNAArtificialprobe 18gggttcccta
agggttggac tcaggtgtga ggggagctgt gacaaggaag aacctcc
571932DNAArtificialprobe 19ctgaggaaac tgcctcttct tccaggtcta tt
322053DNAArtificialprobe 20tgggaaacct tcactctcag cccagccggg
tctagattgg atcttgctgg cgc 532174DNAArtificialprobe 21gggttcccta
agggttggac gtgttcttat ctaggatact ccaaggccaa tttctccatc 60ggtcccatga
tgct 742252DNAArtificialprobe 22tgcccttgca gggacctaca gatgctacgg
ttctgttctc tcgtcagacg tg 522369DNAArtificialprobe 23gggttcccta
agggttggac ggggcgcggc tgcctgtctg caccggcagc accatgtcgc 60tcatggtca
692432DNAArtificialprobe 24tcagcatggc gtgtgttggt gagtcctgga aa
322567DNAArtificialprobe 25tgaccatgag cgacatggtg ctgccggtgc
agacgggagg ttggtctaga ttggatcttg 60ctggcac 672659DNAArtificialprobe
26gggttcccta agggttggac ctgcttcaga acatggctct ctgctgggga gacacccaa
592754DNAArtificialprobe 27tctgcaggcc catagtgtaa ccctggtgct
ccttcccttc caggactcac caag 542857DNAArtificialprobe 28acatgccagg
atgatgaccg tgggtgacat ggagtctaga ttggatcttg ctggcac
572967DNAArtificialprobe 29gggttcccta agggttggac cgagtaaacc
ggaaaatttt catctgcaca gagaggggac 60gtttaac 673047DNAArtificialprobe
30cacactttgc gcctcattgg agagcacatt gatggggtct ccaaggg
473171DNAArtificialprobe 31caacttctcc atcggtcgca tgacacaaga
cctggcatag cgaatacgtc tagattggat 60cttgctggca c
713272DNAArtificialprobe 32gggttcccta agggttggac taccccatcg
ctcttcatgc tggatcattc actctgcatc 60ccaatgacaa tg
723364DNAArtificialprobe 33agaagaaagt ctggacactc tcacctatga
tcacgatgtc cagagggtca ctgggagctg 60acac 643453DNAArtificialprobe
34ctgatagggg gagtgagtaa cagaaccgta gtctagattg atcttgctgg cac
533578DNAArtificialprobe 35gggttcccta agggttggac acagggccca
tgaaaaggct gttccagaat attatgttgt 60agagctcagg gacaggca
783667DNAArtificialprobe 36ccccatcttc cttttacaga ctgaagttgt
taaacccaag ataagaatga cactgaagaa 60tcacata 673750DNAArtificialprobe
37tcctggaggc accacagggc ttggccagtc tagattgatc ttgctggcgc
503870DNAArtificialprobe 38gggttcccta agggttggac tgcacagttg
gatcactgcg ttttcacaca gagaaaaatc 60actcgccctt
703966DNAArtificialprobe 39ctcagaggcc caagacaccc ccaacagata
tcatcgtgta cacggaactt ccaaatgctg 60agccct 664067DNAArtificialprobe
40gatccaaagt tgtctcctgc ccatgagcac cacagtcagg ccttgtctag attgatcttg
60ctggcgc 674181DNAArtificialprobe 41gggttcccta agggttggac
ttgttcatca gaatcctgga gagagggaaa tgctgagtga 60gggagggtgc tcacattttt
c 814264DNAArtificialprobe 42aggactcttt gggaataaca ctagccacga
ggctgggccg aggagcacct acctcgctgt 60tcac 644374DNAArtificialprobe
43ttctgttccc tgcaggctct tggtccatta cagcagcatc tgtaggaaga cgtctagatt
60gatcttgctg gcgc 74
* * * * *
References