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United States Patent
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6,197,509
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Xu
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March 6, 2001
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Method of analyzing DNA using contiguous repeats
Abstract
A novel, highly polymorphic DNA marker based on the
pentanucleotide repeat (CCTTT/GGAAA).sub.n has been identified in the
human inducible nitric oxide synthase (iNOS) gene.
Twelve different alleles, having between 7 and 18 repeats, have been
identified. The repeat is highly polymorphic in the human population
and so lends itself to use as a microsatellite marker
with uses in, for example, forensic medicine, population studies,
family linkage studies and disease diagnosis.
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Inventors:
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Xu; Weiming (Cambridge, GB)
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Assignee:
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Medical Research Council (London, GB)
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Appl. No.:
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155942
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Filed:
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May 13, 1999
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PCT Filed:
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April 4, 1997
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PCT NO:
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PCT/GB97/00949
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371 Date:
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May 13, 1999
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102(e) Date:
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May 13, 1999
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PCT PUB.NO.:
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WO97/38130
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PCT PUB. Date:
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October 16, 1997
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Foreign Application Priority Data
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Current U.S. Class:
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435/6; 435/91.2; 536/23.2; 536/23.5; 536/24.31;
536/24.33
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Intern'l Class:
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C12Q 001/68; C12P 019/34; C07H 021/04
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Field of Search:
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435/6,91.2 536/24.31,24.33,23.2,23.5
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References Cited [Referenced
By]
U.S. Patent Documents
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5882908
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Mar., 1999
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Billiar et al.
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435/189.
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Other References
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Spitsin, S.V. et al. Molecular Medicine 2(2):226-235, Mar.
1996.
Spitsin, S.V. et al. GenBank Accession No. Z49251, Jan.
1997.
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Primary Examiner: Myers; Carla J.
Assistant Examiner: Johannsen; Diana
Attorney, Agent or Firm: Pillsbury Madison & Sutro
Claims
What is claimed is:
1. A method of analyzing a genetic marker in an individual, said
method comprising determining the number of contiguous repeats of
sequence (CCTTT/GGAAA) in the inducible nitric oxide synthase (iNOS)
gene in a biological sample obtained from said individual.
2. The method of claim 1 wherein said determining comprises the steps
of contacting said sample with a pair of oligonucleotide primers,
carrying out a polymerase chain reaction with said primers to amplify
the contiguous repeats of sequence (CCTTT/GGAAA) in the iNOS gene and
determining the number of contiguous repeats of sequence
(CCTTT/GGAAA) in resulting products to determine the number of
contiguous repeats of sequence (CCTTT/GGAAA) in the iNOS gene.
3. The method of claim 2 wherein the number of contiguous repeats in
said products is determined by nucleotide sequencing.
4. The method according to claim 2 wherein said primer pair includes
a forward primer having the sequence 5'-ACCCCTGGAAGCCTACAACTGCAT-3'
(SEQ ID NO:1) and a reverse primer having the sequence
5'-GCCACTGCACCCTAGCCTGTCTCA-3' (SEQ ID NO:2).
5. The method according to one of claims 2-4, which further comprises
contacting said sample with a second pair of oligonucleotide primers
and determining the number of repeats of a second sequence.
6. The method according to claim 5, wherein said second sequence is
the polymorphic trinucleotide repeat (ATT/TAA) of neuronal nitric
oxide synthase (NOS1) gene.
7. The method according to claim 5 wherein said second pair of
oligonucleotide primers includes a forward primer having the sequence
5'-GAAATTGGTCATAGTGGGAATG-3' (SEQ ID NO:3) and a reverse primer
having the sequence 5'-GTGTTGGTGAACCAACCCTCCTAA-3' (SEQ ID NO:4).
8. The method of claim 2 wherein the number of contiguous repeats in
said products is determined by gel electrophoresis.
9. A method of determining the number of contiguous repeats of
sequence (CCTTT/GGAAA) in the inducible nitric oxide synthase (iNOS)
gene of a sample of DNA, said method comprising contacting said
sample with a pair of oligonucleotide primers, carrying out a
polymerase chain reaction with said primers to amplify the contiguous
repeats of sequence (CCTTT/GGAAA) in the iNOS gene and determining
the number of contiguous repeats of sequence (CCTTT/GGAAA) in
resulting products, thereby determining the number of contiguous
repeats of sequence (CCTTT/GGAAA) in the iNOS gene.
10. The method of claim 9 wherein the number of contiguous repeats in
said products is determined by nucleotide sequencing.
11. The method according to claim 9 wherein said primer pair includes
a forward primer having the sequence 5'-ACCCCTGGAAGCCTACAACTGCAT-3'
(SEQ ID NO:1) and a reverse primer having the sequence
5'-GCCACTGCACCCTAGCCTGTCTCA-3' (SEQ ID NO:2).
12. The method according to one of claims 9-11, which further
comprises contacting said sample with a second pair of
oligonucleotide primers and determining the number of repeats of a
second sequence.
13. The method according to claim 12, wherein said second sequence is
the polymorphic trinucleotide repeat (ATT/TAA) of neuronal nitric
oxide synthase (NOS1) gene.
14. The method according to claim 12 wherein said second pair of
oligonucleotide primers includes a forward primer having the sequence
5'-GAAATTGGTCATAGTGGGAATG-3' (SEQ ID NO:3) and a reverse primer
having the sequence 5'-GTGTTGGTGAACCAACCCTCCTAA-3' (SEQ ID NO:4).
15. A primer pair comprising a forward primer having the sequence
5'-ACCCCTGGAAGCCTACAACTGCAT-3' (SEQ ID NO:1) and a reverse primer
having the sequence 5'-GCCACTGCACCCTAGCCTGTCTCA-3' (SEQ ID NO:2),
wherein said primer pair specifically amplifies contiguous repeats of
sequence (CCTTT/GGAAA) in the inducible nitric oxide synthase (iNOS)
gene.
16. a primer pair comprising a forward primer having the sequence
5'-GAAATTGGTCATAGTGGGAATG-3' (SEQ ID NO:3) and a reverse primer
having the sequence 5'-GTGTTGGTGAACCAACCCTCCTAA-3' (SEQ ID NO:4),
wherein said primer pair specifically amplifies a polymorphic
trinucleotide repeat (ATT/TAA) of neuronal nitric oxide synthase
(NOS1) gene.
17. The method of claim 9 wherein the number of contiguous repeats in
said products is determined by gel electrophoresis.
Description
FIELD OF THE INVENTION
This invention concerns analysis of DNA particularly for examining
genetic markers, which is useful in, for example, forensic medicine,
population studies, family linkage studies and disease diagnosis.
BACKGROUND TO THE INVENTION
It is known that there are simple nucleotide sequences in the human
genome that can occur in different numbers of repeats in different
individuals, giving rise to a range of different alleles or variants
of different length that can be used as genetic markers to typify the
DNA of an individual.
Tandem repeat minisatellite and microsatellite regions in vertebrate
DNA frequently show high levels of allelic variability in the number
of repeat units. These highly informative genetic markers have found
widespread applications in population genetics, forensic science,
medicine and other natural scientific studies. For example, these
markers can be used for linkage analysis, determination of kinship in
paternity and immigration disputes and for individual identification
in forensic medicine. In a minisatellite system, a core DNA sequence
unit is usually 15 or more base pairs. To date most studies and
applications of such systems have relied on Southern blot estimation
of allele length, which requires at least 50 ng of relatively
undegraded DNA. It is often very difficult to extract such large
amounts of DNA from many forensic samples such as blood and semen
stains.
Microsatellites, on the other hand, are short tandemly repeated (STR)
polymorphic DNA sequences which are most commonly in the form of
dinucleotide repeats such as (dC-dA)n, but can also be trinucleotide
and tetranucleotide repeats. For a further discussion, see Pena. S.
D. J. and Chakraborry, R. (1994). Paternity testing in DNA era.
Trends in Genetics Vol.10, 204-209. Microsatellites can be amplified
using the polymerase chain reaction (PCR) and the resulting ampileons
normally range from 80-800 base pairs (bps) in length and so are well
suited to processing in automated sequencing machines which are now
widely used for gene scanning and typing. (See Read, P. W. et al
(1994), Chromosome-specific microsatellite sets for fluorescence
based, semiautomatic genome mapping. Nature Genet. 7,390-395.) To
date, most microsatellite polymorphisms have been based upon
dinucleotide repeats. Because of the very small size difference
between adjacent alleles, some of the results can be difficult to
interpret. Tri and tetranucleotide repeats are easier to use but
occur less frequently in the human genome. Expansion of trinucleotide
repeat sequences has also been implicated in a number of genetic
diseases, including Huntingdon's disease, fragile X syndrome and
myotoaic dystrophy.
The present invention is based on the discovery in the human
inducible nitric oxide synthase (iNOS) gene of a pentanucleotide
repeat (CCTTT/GGAAA)n. The repeat is located approximately 2.8 kb 5'
end of upstream promotor region of the iNOS gene on 17q11.1-q11.2.
Investigations have shown this pentanucleotide repeat (which is
referred to for convenience as Xu-1) occurs in widely varying numbers
in different individuals; so far, 12 different variants or alleles
have been detected, having between 7 and 18 contiguous Xu-1 repeats.
The different alleles are referred to as A7, A8 . . . A18. Because
the Xu-1 repeat is highly polymorphic in the human population, the
repeat leads itself to use as a microsatellite marker with uses in,
for example, forensic medicine, population studies, family linkage
studies and disease diagnosis.
SUMMARY OF THE INVENTION
In one aspect the present invention provides an a method of analysing
a sample of DNA to determine the number of contiguous repeats of the
sequence (CCTTT/GGAAA) in the iNOS gene.
The number of repeats is typically in the range 7 to 18.
By analysing DNA in this way, a determination can be made of the
number of Xu-1 repeats in a particular DNA sample, that is which
allele or alleles (usually one or more of A7 to A18) are present
The sample may be, for example, a sample of blood, semen, saliva,
buccal cells or any other suitable biological material.
Because the Xu-1 repeat is a pentanucleotide repeat, it is easier to
distinguish adjacent alleles simply on the basis of size than is the
case for smaller repeating units. Experiments have also shown that
there is considerable variation in the two alleles (one from each
chromosome) of different individuals. The heterozygosity has been
calculated as 0.841. The Xu-1 repeat is therefore highly polymorphic
and hence has significant valve as a genetic marker (the Xu-1 marker)
that is easy to use.
Samples are conveniently analysed by use of the polymerase chain
reaction (PCR), enabling the method of the invention to be performed
on small quantities of sample. A pair of PCR primers has been
designed for this purpose, generating products in the range about 170
to 225 base pairs (bp) in size. The forward primer is
5'-ACCCCTGGAAGCCTACAACTGCAT-3' (Seq. ID No. 1). The reverse primer is
5'-GCCACTGCACCCTAGCCTGTCTCA-3' (Seq. ID No. 2).
The resulting products may be sequenced to determine the number of
Xu-1 repeats. Alternatively, fragment length can simply be
determined, eg by running on an electropheretic gel, enabling
calculation of the number of Xu-1 repeats.
Heterozygosity can be increased substantially by using the Xu-1
marker in conjunction with another genetic marker. For example, good
results have been obtained using the Xu-1 marker with a known
microsatellite marker based on the polymorphic trinucleotide repeat
(ATT/TAA) present in the neuronal nitric oxide synthase (NOS1) gene
in repeat numbers ranging from 5 to 13. PCR primers have been
designed for use with the NOS1 marker to generate products in the
range 110 to 138 bp (ie distinct in size from the Xu-1 marker
products), that can be used under the same PCR conditions as the Xu-1
primers. The forward primer is 5'-GAAATTGGTCATAGTGGGAATG-3' (Seq. ID
No. 3). The reverse primer is 5'-GTGTTGGTGAACCAACCCTCCTAA-3' (Seq. ID
No. 4).
PCR reactions for the 2 markers can thus be run together, and by
using different labels (eg green and blue) for the primers, the PCR
products can be detected simultaneously on the same gel. By using
these 2 markers together, heterozygosity is incresed to about
99%.
Experiments have shown that there are significant differences in the
distribution of alleles with different numbers of the Xu-1 repeat in
different ethnic groups. The Xu-1 marker may therefore be of value in
population studies, immigration disputes, paternity determination and
forensic studies.
Furthermore, because the Xu-1 marker is located in the 5' end of
human iNOS gene, which has been implicated in certain common human
diseases, such as Alzheimer's disease, hypertension, diabetes and
cancers, the marker can be used in allelic association studies and
mutation analysis for the diseases. For example, a strong allelic
association has already been detected between the repeat number and
the senile dementia Lewy body (SDLT) variant of Alzheimer's disease,
which represents about one quarter of all cases of Alzheimer's
disease. Mutations in flanking sequences have also been detected in
some cases of colon cancer. The iNOS gene is also involved in tissue
transplantation. The polymorphic marker described in this invention
also could be used in genotype typing for tissue transplantation.
The invention will be further described, by way of illustration, in
the following Examples and by reference to the accompanying
Figures.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1A is a restriction map of 5' end upstream region of the human
inducible nitric oxide synthase (iNOS) gene, including the Xu-1
repeat region;
FIG. 1B shows the sequence of a 383 base pair Pst1 fragment (the
upper strand of which is Seq. ID No. 5) included in FIG. 1, with Pst1
sites underlined and 11 contiguous Xu-1 repeats marked by boxes;
FIG. 2 shows an electrophoretic gel showing different Xu-1 alleles
from different individuals;
FIG. 3 is a family tree and electrophoretic gel showing different
Xu-1 alleles from different family members;
FIGS. 4A-4D shows various family trees, with Xu-1 allele data;
FIG. 5 shows an electrophoretic gel showing different Xu-1 alleles in
combination with another microsatellite marker for different
individuals;
FIG. 6 is a chart showing the distribution of different Xu-1 alleles
(A8 to A15) for a number of normal individuals, autopsied patients
with the senile dementia Lewy body (SDLT) variant of Alzheimer's
disease and non-Lewy body type Alzheimer's patents (AD) with the x
axis showing % of chromosomes and the y axis showing the allele
numbers; and
FIG. 7 is a chart similar to FIG. 6 showing the distribution of
different Xu-1 alleles (A8 to A18) in Caucasian, Black, Chinese and
Gujerati Asian populations.
EXAMPLE 1
The new pentanucleotide repeat, (CCTTT/GGAAA)n on which the present
invention is based was identified from a comid clone. pCOS4
(described by Xu, W., Charles, I., Moncada, S., Gorman P., Sheer, D.,
Liu, L., and Emson, P. C. (1994) in Mapping of the genes encoding
human inducible and endothelial nitric oxide synthase (NOS2 and NOS3)
to the pericentric region of chromosome 17 and to chromosome 7,
respectively. Genomics 21, 419-422), which contained a 35 kb human
genomic insert, which has also been shown to contain the human
inducible mitric oxide synthase (iNOS) gene coding region and its
promoter region. A restriction map of the 5' upstream end of the gene
is shown in FIG. 1A. In order to clone human promoter region of the
inducible nitric oxide synthase gene, the cosmid was shot-gun cloned
with Pst1 and HindIII restriction enzymes into pBluescript SK vector.
Subclones were then sequenced using an ABI automatic sequencer with
M13 universal and reverse primers. One of the Pst1 subclones, clone
number 512, has been shown by sequencing studies to contain eleven
perfect contiguous pentanucleotide repeats. (CCTTT/GGAAA)11, that is
a stretch of fifty-five bases pairs of human genome, located 2.8 kb
of 5' end of the major transcription initiation site of the human
inducible nitric oxide synthase gene. The sequence of this 383 base
pair fragment (Seq. ID No. 5) is shown in FIG. 1B, with 11
pentanucleotide repeats marked in boxes. It will be seen that the
repeats and flanking regions constitute polypurines in one strand and
polypyrimidines is the other strand spanning over 130 base pairs,
which is highly unusual in a microsatellite because of the
instability of such sequences.
EXAMPLE 2
A pair of specific oligonucleotide primers were designed to use the
polymerase chain reaction (PCR) directly to amplify the polymorphic
pentanucleotide repeat from the genomic DNA from a range of human DNA
samples.
The DNA can be isolated from either blood or buccal cells of the
saliva or any other biological sources which containing nuclei. The
DNA extraction can be done in all cases using standard SDS-Proteinase
K-Phenol procedure. (Sambrook, J., Fritsch, E. F. & Maniatis, T.
(1989). Molecular cloning. Laboratory manual Cold Spring Harbor
Laboratory Press, New York.)
A pair of flanking PCR primers designed to amplify the genomic DNA
from human blood genomic DNA were as follow: The forward primer is
5'-ACCCCTGGAAGCCTACAACTGCAT-3' (Seq. ID No. 1). The reverse primer is
5'-GCCACTGCACCCTAGCCTGTCTCA-3' (Seq. ID No. 2). The primers are
double underlined in FIG. 1B. The forward primer is 5' end labelled
with fluorescent dye 6-carboxyfluorescein (6-Fam) (Oswel DNA
Services, University of Southampton, Southampton SO16 7PX) or Hex
phosphotamidites (Applied Biosystems). Primers were synthesised,
labelled and HPLC-purified using standard methods (Oswel DNA
Services, Southampton).
The PCR protocol is as follows:
i) Reagent Mixture
The following reagents were mixed in a labelled 0.5 ml
double-snap-cap microcentrifuge tube:
Reagent Amount
Template 1 .mu.l 50-200 nm Genomic DNA
10 .times. PCR buffer II (Cetus, 5 .mu.l
N808-0010, E8064)
dNTP Mix (2.5 mM each, 5 .mu.l
Pharmacia, 27-2035-01)
25 mM MgCl.sub.2 (Cetus, N808-0010, 3 .mu.l
E 0843)
Forward primer (200 .mu.g/ml, 1 .mu.l
6 Fam labelled)
Reverse primer (200 .mu.g/ml) 1 .mu.l
dH.sub.2 O (distilled or deionized water) 33.5 .mu.l
AmpliTaq DNA polymerase 0.5 .mu.l (2.5 Units)
(Cetus, N801-0060)
Final Reaction Volume 50 .mu.l
(AmpliTaq is a Trade Mark of Roche Molecular Systems, Inc.)
(dNTP=deoxynucleotide triphosphate. Taq DNA polymerase=DNA polymerase
isolated from Thermus aquaticus.)
The reaction mixture was overlaid with one drop of mineral oil (Sigma
No. M5904) (approximately by 40 .mu.l).
ii) PCR Reaction
PCR is carried out using a Perkin Elmer Cetus Model 480 (or
equivalent machine) as follows.
1. Place the tubes in a thermal cycler.
2. Immediately after placing the tubes in the thermal cycler, begin
thermal cycling as follows:
a) preheat to 96.degree. C.
b) 96.degree. C. for 30 seconds
c) 30 cycles as follows: 94.degree. C. for 1 min., annealing at
50.degree. C. for 1 min., and polymerising at 72.degree. C. for 1
min.
d) 72.degree. C. for 10 min.
3. Rapid thermal ramp to 4.degree. C. and hold.
The sizes of the PCR products range from about 170 bp to 225 bp.
dependent on the number of pentanucleotide repeat units.
iii) Electrophoresis
The PCR products are then loaded directly on an Applied Biosystems
Model 373A DNA Sequencer using 6%-urea polyacrylamide gel. Running
conditions are at 2000V, 26 watts for 4 to 12 hours. The GENESCAN
option is used to start running the gel. (Genescan is a Trade Mark of
Applied Biosystems, Inc.)
Preparing and loading the samples was carried out as follows:
1. Prepare a mixture of the following reagents:
5 .mu.l detonized formamide
0.5 .mu.l Rox labelled DNA marker (GENESCAN-2500)
2. Add 4 .mu.l of this mixture to each tube and agitate vigorously.
Centrifuge the solution briefly.
3. When the gel is ready for loading, heat the samples at 90.degree.
C. for 2 minutes to denature, then transfer them immediately onto
ice.
4. Load the samples onto an Applied Biosystems 373A DNA Sequence
according to the instructions in the User's Manual.
iv) Genescan and Analysis
The GENESCAN 672 software is used to collect and analyse the data
automatically. This software can be used not only to collect
electroophoretic data across all 24 or 36 lanes, but also accurately
to identify and analyse the different lanes of the fragments. The
internal standard, Rox-GENESCAN 2500, permits accurate and precise
base identification and accurate sizing of the fragments. If an
automatic DNA sequencer is not available, other methods such as use
of radioactive labels can be used instead to detect the PCR
products.
v) Sequence Analysis
The PCR products can also be cloned into the Bluescripts pKS--vector
using the T-vector cloning system (Stratagene, Cambridge, UK) and
sequenced by TaqDyeDeoxy terminator cycle sequencing with an Applied
Biosystems Model 373A DNA Sequencer, using vector universal and
reverse primers. The products can also directly sequenced using the
PCR DNA gel purification system from Qiegen, following the
manufacturers instructions, with 3 pmol of Primer A or B.
EXAMPLE 3
Using the primers and techniques described in Example 2. DNA from 36
unrelated individuals was examined. The resulting electrophoretic gel
is shown in FIG. 2. A series of (red) bands (not visible in the
Figure) show internal DNA size markers (Genescan 2500, Rox), as
indicated on the left of the Figure. The brighter (blue) bands across
the middle of the gel are produced by 6-Fam fluorescently labelled
primer A, and these show the presence of different repeat lengths,
demonstrating polymorphism.
EXAMPLE 4
Data was obtained in a similar manner from 3 generations of a
Caucasian (CEPH (Centre Etude Polymorpisme Humain)/Amish) family
(pedigree 884). The results are shown in FIG. 3 in the form of a
family tree and gel data showing Xu-1 allele data. The results are
fully consistent and show Mendelian co-dominant inheritance. Other
family groups have been similarly analysed and all show the same
Mendelian co-dominant inheritance manner of this locus. for example,
FIG. 4 shows the genotypes of four large CEPH families (pedigrees
884, 1424, 1341 and 1349).
EXAMPLE 5
By using a probe for the Xu-1 repeat in conjunction with a probe for
another genetic marker, more detailed specific genetic information
can be obtained about an individual, that is, a more detailed
"genetic fingerprint" can be obtained, resulting in increased
heterotygosity and hence usefulness of the results. Experiments were
carried out using probes for the Xu-1 repat as described above, in
conjunction with probes for a known trinucleotide repeat (ATT/TAA)
present in the neuronal nitric oxide synthase (NOS1) gene in repeat
numbers ranging from 5 to 13. The NOS1 repeat is described by Chung,
E., Curus D., Chen, G., Marsdea, P. A., Twells. R., Xu, W. and
Gardener, M. (1996) in Genetic evidence for the neuromal nitric oxide
synthase gene as a susceptibility for infantile pyloric stenosis, Am.
J. Hum. Genet. 58, 363-370.
For this purpose, PCR primers were designed for use with the NOS1
marker to give products in the range 110 to 138 bp, fluorescently
labelled green with 5-Hexdye. The forward primer is
5'-GAAATTGGTCATAGTGGGAATG-3' (Seq. ID No. 3). The reverse primer is
5'-GTGTTGGTGAACCAACCCTCCTAA-3' (Seq. ID No. 4). This pair of the
primers can be used under exactly the same PCR conditions as the
primers for the Xu-1 repeat labelled with Famdye, so two PCR
reactions can be performed at the same time.
FIG. 5 shows a Genescan gel obtained by this procedure, with the
upper (blue) bands showing the Xu-1 repeats and the lower (green)
bands the NCS1 repeats with results aligned for each sample. The
combined heterozygeosity is about 99%.
EXAMPLE 6
Because the Xu-1 repeat of the invention is located at the 5' end of
the human iNOS gene, which has been implicated in certain diseases
including Alzheimer's disease, experiments were carried out on 112
deceased demented patients diagnosed by autopsy as having Alzheimer's
disease, both the senile dementia Lewy body (SDLT) variant of
Alzheimer's disease (22 patients) and non-Lewy body type (AD) (90
patients). For comparison, results were also obtained from 101 normal
Caucasian individuals. The DNA for all subjects was obtained from the
Cambridge Brain bank and was approved by the local ethics committee.
Experiments were performed generally as described in Example 2.
The results are shown graphically in FIG. 6. The results show that
most alleles have similar frequency in patients with Alzheimer's
disease and non-demented controls. However, in samples from the SDLT
patients, two smaller alleles, A8 and A9 (having 8 and 9
pentanucleotide repeats, respectively) are over-represented (16%,
compared to normal 3% and AD 4%) and the A11 allele is
under-represented (2% compared to normal 19%). Using the counting
program from Linkage Utility, the p-value is calculated to be 0.0151
(6 degree freedom), which is much lower than required value 0.05
(5%). The results suggest that certain combinations of the Xu-1
pentanucleotide repeat variant in iNOS gene promoter, namely high A11
allele and/or low A8 and A9 alleles, may be associated with
development of SDLT. This information may be of diagnostic or
predictive value.
EXAMPLE 7
Experiments were carried out in similar manner on 271 individuals (ie
542 chromosomes) of Caucasian, Black (Afro Caribbean and Afro
American), Chinese and Gujerati Asian ethnic origin and the
distribution of different numbers of the xu-1 repeat were analysed by
ethnic group. The results are shown in tabular form in Tables 1 and
2, and graphically in FIG. 7. The degree of polymorphism is
characterised by two indices: Heterozygosity (Heter) and polymorphism
information content (PIC). For formulas and explanation, see
Botstein, D., White, R. L. Skolnick, M. and Davis, R. W. Construction
of a genetic linkage map in man using restriction fragment length
polymorphisms. Am. J. Hum. Genet. 32:314-331. (1980). The overall
heterozygosity was 0.841.
There are significant differences in allelic frequency of
distribution apparent between the different ethnic groups. These
results may therefore be of forensic value.
TABLE 1
(CCTTT)n Size (bp)* Caucasian Black Chinese Gujeratis Combined
8 175 0 1(0.006) 0 0 1(0.002)
9 180 7(0.035) 9(0.058) 0 5(0.035) 21(0.039)
10 185 25(0.124) 19(0.123) 13(0.31) 20(0.139) 77(0.142)
11 190 38(0.188) 14(0.09) 4(0.10) 23(0.16) 79(0.146)
12 195 59(0.292) 37(0.24) 9(0.21) 29(0.20) 134(0.247)
13 200 47(0.233) 28(0.181) 2(0.05) 23(0.16) 100(0.184)
14 205 20(0.10) 19(0.123) 6(0.14) 17(0.22) 62(0.114)
15 210 5(0.035) 25(0.162) 3(0.07) 20(0.14) 53(0.098)
16 215 2(0.005) 2(0.013) 1(0.02) 5(0.035) 9(0.017)
17 220 0 0 3(0.07) 1(0.007) 4(0.007)
18 225 0 0 1(0.02) 1(0.007) 2(0.004)
Total 202 154 42 144 542
No. of alleles 8 9 9 10 11
Heterozygosity 0.802 0.846 0.835 0.859 0.841
PIC 0.769 0.82 0.793 0.835 0.81901
*Due to flanking sequences and running condition size varies +-2bp.
TABLE 2
Population A: Population B: d.f. X.sup.1 P value
Caucasian: Black 7 30.05 0.000083
Caucasian: Chinese 7 34.89 0.000012
Caucasian: Gujeratis 7 27 0.000235
Black: Chinese 7 32.16 0.000038
Black: Gujeratis 7 8.46 0.2938
Chinese: Gujeratis 7 14.65 0.0407
* * * * *