U.S. patent application number 11/151453 was filed with the patent office on 2005-10-20 for diagnosis of a person's risk of developing atherosclerosis or diabetic retinopathy based on leucine 7 to proline 7 polymorphism in the prepro-neuropeptide y gene.
This patent application is currently assigned to Hormos Medical Oy. Invention is credited to Karvonen, Matti, Koulu, Markku, Pesonen, Ullamari, Uusitupa, Matti.
Application Number | 20050234008 11/151453 |
Document ID | / |
Family ID | 23122742 |
Filed Date | 2005-10-20 |
United States Patent
Application |
20050234008 |
Kind Code |
A1 |
Koulu, Markku ; et
al. |
October 20, 2005 |
Diagnosis of a person's risk of developing atherosclerosis or
diabetic retinopathy based on leucine 7 to proline 7 polymorphism
in the prepro-neuropeptide Y gene
Abstract
The invention relates to methods for diagnosing a person's
susceptibility for having an increased risk for the development of
atherosclerosis and a diabetic person's susceptibility for having
an increased risk for the development of diabetic retinopathy. The
invention relates further to methods for treating persons diagnosed
for having increased risk for the development of said diseases, in
order to prevent the development of said diseases. The invention
also concerns methods to investigate or screen pharmaceuticals or
genetic aims useful in the treatment of said diseases, by using an
animal model including a transgenic animal.
Inventors: |
Koulu, Markku; (Turku,
FI) ; Karvonen, Matti; (Turku, FI) ; Pesonen,
Ullamari; (Turku, FI) ; Uusitupa, Matti;
(Kuopio, FI) |
Correspondence
Address: |
ROTHWELL, FIGG, ERNST & MANBECK, P.C.
1425 K STREET, N.W.
SUITE 800
WASHINGTON
DC
20005
US
|
Assignee: |
Hormos Medical Oy
Turku
FI
|
Family ID: |
23122742 |
Appl. No.: |
11/151453 |
Filed: |
June 14, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11151453 |
Jun 14, 2005 |
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09937899 |
Sep 28, 2001 |
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09937899 |
Sep 28, 2001 |
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PCT/FI00/00260 |
Mar 29, 2000 |
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09937899 |
Sep 28, 2001 |
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09291994 |
Apr 15, 1999 |
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6312898 |
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Current U.S.
Class: |
514/44A ;
536/23.1 |
Current CPC
Class: |
A61P 3/10 20180101; C12Q
2600/156 20130101; A61P 43/00 20180101; A61P 9/10 20180101; A61P
27/02 20180101; C12Q 1/6883 20130101; A61K 31/00 20130101 |
Class at
Publication: |
514/044 ;
536/023.1 |
International
Class: |
A61K 048/00; C07H
021/02 |
Claims
1. A method for treating a person, diagnosed for having an
increased risk for the development of atherosclerosis on the basis
of a polymorphism in the signal peptide part of the human
preproNPY, said polymorphism comprising the substitution of proline
for the position 7 leucine in the signal peptide part of said
preproNPY, for the prevention of developing atherosclerosis,
comprising administering to said person an effective amount of an
agent counteracting the influence of the mutated NPY gene.
2 The method according to claim 1, wherein said agent is a
pharmaceutical aimed to modulate gene expression of normal or
mutated NPY gene.
3. A method for treating a diabetic person, diagnosed for having an
increased risk for the development of diabetic retinopathy on the
basis of a polymorphism in the signal peptide part of the human
preproNPY, said polymorphism comprising the substitution of proline
for the position 7 leucine in the signal peptide part of said
preproNPY, for the prevention of developing diabetic retinopathy,
comprising administering to said person an effective amount of an
agent that modulates gene expression of the NPY gene comprising
said polymorphism, wherein said agent is an antisense
oligonucleotide that is complementary to the sequence
5'-acaagcgaccgg-3' (SEQ ID NO:9) and includes a nucleotide
complementary to the nucleotide c at position 10 of SEQ ID
NO:9.
4. The method of claim 2, wherein said oligonucleotide is said
antisense oligonucleotide comprises 7 or 8 nucleotides.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S. patent
application Ser. No. 09/937,899 filed 28 Sep. 2001, which in turn
is a national stage filing under 35 U.S.C. .sctn.371 of
international application No. PCT/FI00/00260, filed 29 Mar. 2000,
which in turn is a continuation of U.S. patent application Ser. No.
09/291,994, filed 15 Apr. 1999, now U.S. Pat. No. 6,312,898.
FIELD OF THE INVENTION
[0002] This invention relates to methods for diagnosing a person's
susceptibility for having an increased risk for the development of
atherosclerosis and a diabetic person's susceptibility for having
an increased risk for the development of diabetic retinopathy. The
invention relates further to methods for treating persons diagnosed
for having increased risk for the development of said diseases, in
order to prevent the development of said diseases. The invention
also concerns methods to investigate or screen pharmaceuticals or
genetic aims useful in the treatment of said diseases, by using an
animal model including a transgenic animal.
BACKGROUND OF THE INVENTION
[0003] The publications and other materials used herein to
illuminate the background of the invention, and in particular,
cases to provide additional details respecting the practice, are
incorporated by reference.
[0004] Neuropeptide Y (NPY) is a member of the pancreatic
polypeptide family and neuromodulator that is secreted widely by
neurons of the central and peripheral nervous systems and it is the
most abundant peptide in the brain and in the heart (1-4). NPY is
the most potent orexigenic neuropeptide and may have tonic
inhibitory action on leptin mediated satiety signal (2-3,5). NPY
stimulates insulin secretion (6) and insulin-induced glucose uptake
in normal rats (7). In contrast, insulin and insulin-like growth
factor II suppress hypothalamic NPY release (8). In animal models
of obesity and Type 2 diabetes, enhanced activity of NPY neurons
due to hypothalamic resistance of insulin inhibition may contribute
to hyperphagia, reduced energy expenditure and obesity (9).
Further, NPY participates in the control on
hypothalamic-pituitary-adrena- l axis (10). In the cardiovascular
system NPY is a vasoconstrictor, it inhibits the release of
norepinephrine and potentiates the norepinephrine response (11).
Interestingly, in experimental diabetes cardiorespiratory responses
to NPY have been shown to be altered (12-13). Further, NPY may have
angiogenic properties (4) that could enhance the development of
atherosclerosis. The widespread effects of NPY are mediated by
several different subtypes of NPY receptors (14). We identified a
rather common leucine7 to proline7 polymorphism (Leu7/Pro) very
recently (15). This polymorphism was found to be associated with
significantly higher serum total- and LDL cholesterol levels
particularly in obese subjects in two independent Finnish and one
Dutch study population. Further, apolipoprotein B levels were
elevated in non-diabetic subjects with Leu7/Pro-polymorphism in one
of these populations (15). Although the biochemical and
physiological link between cholesterol metabolism and NPY is
currently not known, the Leu7/Pro-polymorphism of NPY gene should
be considered as a new genetic marker for high cholesterol levels
in obese subjects.
SUMMARY OF THE INVENTION
[0005] According to one aspect, this invention concerns a method
for diagnosing a person's susceptibility for having an increased
risk for the development of atherosclerosis, said method comprising
determining whether said subject has a polymorphism in the signal
peptide part of the human preproNPY, said polymorphism comprising
the subsitution of the position 7 leucine for proline in the signal
peptide part of said preproNPY, said polymorphism being indicative
of an increased risk for the development of atherosclerosis.
[0006] According to another aspect, the invention concerns a method
for diagnosing a diabetic person's susceptibility for having an
increased risk for the development of diabetic retinopathy, said
method comprising determining whether said subject has a
polymorphism in the signal peptide part of the human preproNPY,
said polymorphism comprising the subsitution of the position 7
leucine for proline in the signal peptide part of said preproNPY,
said polymorphism being indicative of an increased risk for the
development of diabetic retinopathy.
[0007] According to a third aspect, the invention concerns a method
for treating a person, diagnosed for having an increased risk for
the development of atherosclerosis, or for treating a diabetic
person, diagnosed for having an increased risk for the development
of diabetic retinopathy, for the prevention of developing any of
said diseases, comprising administering to said person an effective
amount of an agent counteracting the influence of the mutated NPY
gene.
[0008] According to a fourth aspect, the invention concerns a
method for treating a person, diagnosed for having an increased
risk for the development of atherosclerosis, or for treating a
diabetic person, diagnosed for having an increased risk for the
development of diabetic retinopathy, for the prevention of
developing any of said diseases, comprising subjecting the person
to specific gene therapy aimed to repair the mutated NPY signal
peptide sequence.
[0009] According to a fifth aspect, the invention concerns a method
to investigate or screen pharmaceuticals or genetic aims useful in
the treatment of atherosclerosis or diabetic retinopathy, by using
an animal model including a transgenic animal which carries a human
DNA sequence comprising a nucleotide sequence encoding a
prepro-neuropeptide Y (preproNPY) or part thereof encoding mature
human NPY peptide, where the leucine amino acid in position 7 of
the signal peptide part of said preproNPY i) is unchanged or ii)
has been replaced by proline.
[0010] According to a sixth aspect, the invention concerns a method
to investigate or screen pharmaceuticals or genetic aims useful in
the treatment of atherosclerosis or diabetic retinopathy, by using
an animal model including a transgenic animal, which carries a DNA
sequence comprising a nucleotide sequence encoding otherwise normal
mouse NPY sequence or part thereof encoding mature mouse NPY
peptide, but in which the nucleotide sequence encoding the mouse
signal peptide is replaced by human signal peptide sequence
encoding either normal or mutated human signal peptide.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1a illustrates schematically the molecular structure of
the human NPY gene, the preproNPY peptide and the mature NPY
peptide,
[0012] FIG. 1b shows the nucleotide sequence of the human NPY gene.
Upper case indicates exonic sequences and lower case intronic
sequences. Genbank accession numbers are given in parenthesis. The
arrow shows the position in which thymidine (T) of the normal gene
is replaced by cytosine (C) to give the mutant gene. The underlined
sequence in Exon 2 is the sequence encoding the signal peptide of
28 amino acids (Exon 1 is SEQ ID NO:1, exon 2 is SEQ ID NO:2, exon
3 is SEQ ID NO:3 and exon 4 is SEQ ID NO:4), and
[0013] FIG. 1c shows the nucleotide sequence of the human preproNPY
mRNA (SEQ ID NO:5, with the protein sequence set forth in SEQ ID
NO:6). The arrow shows the position in which thymidine (t) of the
normal mRNA is replaced by cytosine (c) to give the mutant
mRNA.
[0014] FIG. 2 shows the predicted secondary structure of preproNPY
mRNA and the predicted structure of the 5' end (1 to 138 bases) of
the full preproNPY mRNA sequence published in GenBank Accession No.
K01911. The secondary structure was predicted by using the MFOLD
program of the Genetics Computer Group of the University of
Wisconsin. Squiggle plot of: osa1.mfold February 7, 19100 12:46.
(Linear) MFOLD of: osa1.seqT: 37.0 Check: 5173 from: 1 to: 138
February 7, 19100 12:43. Length 138 Energy -28.4. The nucleotide
sequence "acaagcgacugg" is the wildtype sequence of SEQ ID
NO:7.
[0015] FIG. 3 shows the predicted secondary structure of mutated
preproNPY mRNA and the predicted structure of the 5' end (1 to 138
bases) of the full mutated preproNPY mRNA sequence published in
GenBank Accession No. K01911. The secondary structure was predicted
by using the MFOLD program of the Genetics Computer Group of the
University of Wisconsin. The mutated base T to C is base number
106. Squiggle plot of: osa2.mfold February 7, 19100 14:11. (Linear)
MFOLD of: osa2.seqT: 37.0 Check: 4340 from: 1 to: 138 February 7,
19100 14:07. Length 138 Energy -26.4 -28.4. The nucleotide sequence
"acaagcgaccgg" is the mutant sequence of SEQ ID NO:7.
DETAILED DESCRIPTION OF THE INVENTION
[0016] Neuropeptide Y (NPY) is a 36-amino-acid neurotransmitter
widely present in the central and peripheral nervous systems. NPY
has multiple actions, which control body energy balance and
cardiovascular function. We have recently demonstrated that the
subjects having Pro7 in the signal peptide of NPY have higher serum
cholesterol and apolipoprotein B levels when compared to
individuals having wildtype (Leu7/Leu7) signal peptide sequence.
The present invention is based on a study of the association of
Leu7 to Pro polymorphism of the NPY gene with common carotid
intima-media-thickness (IMT) assessed by ultrasonography
cross-sectionally from the 10-year follow-up study of newly
diagnosed patients with Type 2 diabetes (81 patients, 41 males,
mean age 67.1 years) and in non-diabetic subjects (105 subjects, 48
males, mean age 65.5 years) who were genotyped for Leu7Pro
polymorphism in preproNPY gene. The carrier frequency of the Pro7
substitution was 9.9% in diabetic patients and 14.3% in control
subjects (p=0.360). The mean common carotid IMT was in non-diabetic
subjects without Leu7Pro polymorphism 1.04.+-.0.02 and with it
1.14.+-.0.04 mm (p=0.156) and in diabetic patients 1.18.+-.0.03 and
1.58.+-.0.21 mm (p=0.004), respectively. In the analysis of
covariance of the entire group the mean common carotid IMT was
independently associated with the Leu7Pro-polymorphism (F=5.165,
p=0.024). The model included age, gender, diabetes, clinical
macrovascular disease, smoking, systolic blood pressure and
LDL-cholesterol. Furthermore, diabetic patients having the Pro7 in
preproNPY had significantly more often diabetic retinopathy
(p=0.04) when compared to patients with the Leu7/Leu7 genotype. The
present study indicates that the presence of Pro7 substitution in
the preproNPY is strongly associated with increased carotid
atherosclerosis in diabetic and non-diabetic subjects, even after
adjustment for known risk factors. Furthermore, this is the first
evidence that Pro7 in the preproNPY increases the risk of type 2
diabetic patients to develop diabetic retinopathy.
[0017] The DNA sequence or the mutant signal peptide or said
peptide associated with any other cleavage product of preproNPY can
be used for screening a subject to determine if said subject is a
carrier of a mutant NPY gene.
[0018] The determination can be carried out either as a DNA analyse
according to well known methods, which include direct DNA
sequencing of the normal and mutated NPY gene, allele specific
amplification using the polymerase chain reaction (PCR) enabling
detection of either normal or mutated NPY sequence, or by indirect
detection of the normal or mutated NPY gene by various molecular
biology methods including e.g. PCR-single stranded conformation
polymorphism (SSCP)-method or denaturing gradient gel
electrophoresis (DGGE). Determination of the normal or mutated NPY
gene can also be done by using restriction fragment length
polymorphism (RFLP)-method, which is particularly suitable for
genotyping large number of samples.
[0019] The determination can also be carried out at the level of
RNA by analysing RNA expressed at tissue level using various
methods. Allele spesific probes can be designed for hybridization.
Hybridization can be done e.g. using Northern blot, RNase
protection assay or in situ hybridization methods. RNA derived from
the normal or mutated NPY gene can also be analysed by converting
tissue RNA first to cDNA and thereafter amplifying cDNA by an
allele spefic PCR-method and carrying out the analysis as for
genomic DNA as mentioned above.
[0020] Alternatively, the determination can be carried out as an
immunoassay where a sample is contacted with an antibody capable of
binding the signal peptide or said peptide associated with any
other cleavage product of preproNPY.
[0021] Antibodies can be raised against normal or mutated preproNPY
or more specifically against normal or mutated signal peptide part
of the NPY. The production of antibodies can be done in
experimental animals in vivo to obtain polyclonal antibodies or in
vitro using cell lines to obtain monoclonal antibodies.
[0022] A person diagnosed for having an increased risk for the
development of atherosclerosis, or a diabetic person, diagnosed for
having an increased risk for the development of diabetic
retinopathy, can be treated for the prevention of developing any of
said diseases administering to said subject an effective amount of
an agent counteracting the influence of the mutated NPY gene. This
can be done by specific gene therapy aimed to repair the mutated
NPY sequence, or by administering pharmacotherapies, which are
aimed to modulate synthesis, release or metabolism of the
endogenous NPY, or to interact in a specific manner at NPY target
sites by modulating effects of NPY with specific NPY receptor
proteins. Currently, five different subtypes of NPY receptors have
been cloned and characterized (Y1-Y5 receptors) and drug molecules
specifically interacting with these NPY receptors have been
synthesized. The pharmacotherapy described is not limited to only
these named receptors or mechanisms, but also covers other NPY
receptors and related mechanisms to be discovered including the
secretion of NPY.
[0023] Counteracting the influence of the mutated NPY gene in a
patient by using an antisense therapy or gene switching or
replacement, which includes targeted correction of disease-related
mutation or site-directed inactivation of the mutant allele by
homologous recombination.
[0024] The antisense therapy refers to methods designed to impair
translation through direct interactions with target messenger RNA
(mRNA). This can be accomplished by applying a targeted
oligonucleotide, which forms Watson-Crick base pairs with the
messenger RNA whose function is to be disrupted. The inhibition of
gene expression by antisense oligonucleotide depends on the ability
of an antisense oligonucleotide to bind a complementary mRNA
sequence and prevent the translation of the mRNA. It is possible to
correct a single mutant base in a gene by using an oligonucleotide
based strategy (Giles et al., 1995; Schwab et al., 1994; Yoon et
al., 1996). A short, 7 or 8 bases, oligonucleotide is enough to
posses an antisense activity and specificity, which depends greatly
on the flanking sequences of the target RNA. Binding should be
enough to promote stable binding and RNase H-mediated cleavage.
[0025] We are counteracting the influence of the mutated NPY gene
by using a short, allele specific oligonucleotide, which includes
the sequence of mutated part: . . . cga ct/cg ggg . . . (mutated
base marked on bold) (SEQ ID NO:8). This can be accomplished by
using oligonucleotides of various lengths, but all recognizing the
mutated base sequence. According to the predicted secondary
structure of the preproNPY mRNAs (FIGS. 2 and 3), the best target
sequence is between -9 and +2 bases around the mutation i.e., a
sequence targeting to 5'-ac aag cga ccg g-3' (SEQ ID NO:9). This
sequence contains `bulbs` which are known to enhance the binding of
oligonucleotide to the target mRNA.
[0026] It is possible to use unmodified oligonucleotides, but to
increase their stability, nuclease resistance, and penetration to
the nucleus, several modifications of oligonucleotide can be used.
A relatively large number of modified pyrimidines have been
synthesized, mainly C-2, C-4, C-5, and C-6 sites, and incorporated
into nucleotides. Also purine analogs can be synthesized and
incorporated into oligonucleotides. The 2' position of the sugar
moiety, pentofuranose ring, is substituted with methoxy, propoxy,
O-alkoxy or methoxyethoxy groups. A new backbone for
oligonucleotides that replace the phosphate or the sugar-phosphate
unit has been made, like C-5 propynylpyrimidine-modified
phosphothioate oligonucleotides. Also chimeric oligonucleotides
with 5'- and 3'-ends are modified with internucleotide linkages,
like methylphosphorothioate, phosphodiester, or methylphosphonate
can be used. A relatively new technique is conformationally
restricted LNA (locked nucleic acid) oligonucleotides and peptide
nucleic acids. Bioengineered ribozymes are structurally different,
but their specificity also relay on the recognition of the targeted
mRNA sequence.
[0027] Gene replacement or gene switching techniques inactivate the
mutated gene sequence and introduce a corrected one. This can be
accomplished by transfecting exogenous gene with normal coding
sequence and blocking mutant coding sequence with antisense
oligonucleotide. Also a technique with only introducing a corrected
normal sequence without disrupting the mutated sequence could be
use. This could be used in heterozygous cells i.e. cell carrying
one normal allele and one mutated allele resulting in an
overexpression of normal alleles. Also homozygous mutant cells
could be treated resulting in a dominant positive-effect i.e. the
normal allele is expressed in higher degree than the mutant
allele.
[0028] Influence of the mutated NPY sequence on the function of NPY
gene can be investigated in transgenic animals. A transgenic animal
can be generated using targeted homologous recombination
methodology. Both normal and mutated sequence of human NPY signal
peptide (or any DNA sequence comprising a nucleotide sequence
encoding a prepro-neuropeptide Y (preproNPY) or part thereof
encoding the amino acid sequence of the mature mouse or human
mature NPY peptide, where either i) the leucine amino acid in
position 7 of the signal peptide part of said preproNPY has been
replaced by proline or ii) the leucine amino acid in position 7 of
the signal peptide part of said preproNPY is unchanged) will be
introduced into the sequence of NPY gene to replace the endogenous
signal peptide sequence. Under these conditions, the endogenous NPY
gene functions otherwise normally, but the synthesis of the
preproNPY is regulated by either normal or mutated human NPY signal
peptide sequence. This transgenic model can be used to investigate
in a very specific manner the physiological importance of the
mutated NPY gene. It also will provide an ideal preclinical model
to investigate and screen new drug molecules, which are designed to
modify the influence of the mutated NPY gene.
[0029] The invention is described more in detail in the following
experiments.
EXPERIMENTAL
[0030] Study Design
[0031] This study was a cross-sectional analysis from the 10-year
examination of a cohort of patients with Type 2 diabetes and
nondiabetic control subjects followed up from the time of
diagnosis, as described earlier in detail (16-22). In brief, the
original study comprised 133 patients with newly diagnosed Type 2
diabetes, aged 45 to 64 years, and 144 nondiabetic control subjects
randomly selected from the population register. The baseline study
was carried out during the years 1979-81 and all subjects were
collected from a defined area in Eastern Finland (16). All the
subjects were invited for the 5- and 10-year follow-up examinations
during the years 1985-86 (17) and 1991-92 (18-19), respectively.
During the 10-year follow-up 36 (27%) diabetic patients and eight
(6%) nondiabetic subjects died, mainly due to cardiovascular
diseases (18). At the 10-year examination, carotid ultrasonographic
examinations (20-21) were performed for 84 (63%) of the original
diabetic and 119 (83%) of the nondiabetic populations and genotype
analysis was made for all these except for three diabetic and one
non-diabetic subject. The study was approved by the Ethics
Committee of the University of Kuopio.
[0032] Subjects and Methods
[0033] The assessment of medical history and cardiovascular
diseases, the use of medication, smoking, blood pressure, body-mass
index (BMI) and waist-to-hip circumference ratio have been
described in detail previously (18-22). The group "macrovascular
disease" refers to subjects with any previously defined evidence of
myocardial infarction, stroke or intermittent claudication. An oral
glucose tolerance test was performed by using a glucose dose of 75
g. The impaired glucose tolerance in control subjects was
classified according to the WHO criteria (23). The collection of
blood specimens and the measurement of serum lipid and lipoproteins
by ultracentrifugation and precipitation methods, apolipoprotein B,
plasma glucose and plasma insulin have been likewise presented
previously (19-22).
[0034] Genotype Analysis
[0035] PreproNPY genotype was determined by restriction fragment
length polymorphism (RFLP) analysis from DNA extracted from the
subjects peripheral blood by an investigator unaware of phenotype.
Briefly, the polymorphism appears as a thymidine(1128) to
cytosine(1128) substitution generating a Bsi EI restriction site,
which was used to genotype the subjects for the Leu7Pro
polymorphism, as described previously (15). The PCR products were
digested by Bsi EI (New England Biolabs, Inc. Beverly, Mass., USA)
and digestions were analyzed by electrophoresis on 2% agarose
gel.
[0036] Assessment of Carotid Atherosclerosis
[0037] The high-resolution B-mode ultrasonographic imaging protocol
was designed to ensure the valid and reliable identification of
arterial carotid references and the definition of near-wall and
far-wall interfaces, as described previously in more detail
(20-21,24). Briefly, the carotid artery was divided into two
segments on the basis of arterial anatomy and geometry. The key
anatomic features defining these segments were the proximal origin
of the bulb (carotid bifurcation) and the tip of the flow divider,
which separates internal from external carotid arteries. In
longitudinal arterial images, the adventitia-media and the
intima-lumen interfaces on the far wall were the specific anatomic
bondaries defining the IMT. Two certified sonographers performed
the carotid ultrasound examinations. A Biosound Phase Two
ultrasound device equipped with a 10-MHz annular array probe was
used. Video-recorded examinations were quantitatively analyzed at a
central laboratory using a computer-assisted reading procedure
(24-25). The mean maximum of the far wall bilaterally was used as
the measurement of the common carotid IMT.
[0038] Statistical Methods
[0039] Our a priori hypothesis was that the subjects having Pro7
substitution in preproNPY have higher mean IMT compared to the
subjects having wild type preproNPY (Leu7/Leu7). Associations of
Leu7Pro polymorphism with continuous variables were calculated
using Student's t-test and for categorized variables by Chi square
test. The association of common carotid IMT with Leu7Pro
polymorphism was further analyzed by analysis of covariance
(ANCOVA) controlling for the effects of selected covariates.
Variables with skewed distribution (eg. carotid IMT, insulin) were
analyzed after logarithmic transformation. P-value equal or less
than 0.05 was considered statistically significant. All statistical
analyses were conducted with procedures from SPSS-Unix.
[0040] Results
[0041] The frequency of C1128 allele frequencies was not
significantly different between non-diabetic (14.3%) and diabetic
(9.9%, p=0.36) groups. The characteristics of non-diabetic and
diabetic subjects for Leu7/Leu7 and Pro7/-groups are presented in
Tables 1a-b. No differences in age, gender, body mass index,
waist-to-hip-ratios, blood pressure levels and the frequencies of
macrovascular disease were found between the genotype groups within
the non-diabetic and diabetic groups. LDL-cholesterol was higher in
non-diabetic subjects with Leu7/Pro-polymorphism than in those
without (p=0.05), as we have reported previously (15). Although
apolipoprotein B levels tended to be higher in Pro7/-group than in
Leu7/Leu7-group, the differences were not statistically
significant. Our previous study included only lean subjects without
any medication known to affect cholesterol metabolism (like
beta-blockers or diuretics) of the present non-diabetic group (15).
In other lipoproteins no evident differences were found, and
interestingly, in diabetic patients there was no association with
serum cholesterol, even when subjects were analyzed according to
median body mass index (data not shown).
[0042] The mean common carotid IMT was about 25% higher in diabetic
patients with Pro7 allele than in those without it (p=0.004) and
the respective increase in IMT was 9% in non-diabetic subjects
(p=0.156). In the analysis of covariance both groups combined
(Table 2) the independent predictors of common carotid IMT were
age, Pro7 allele, diabetes, systolic blood pressure, and
macrovascular disease. Furthermore, those diabetic patients having
the Pro7 substituion in the preproNPY had significantly accelerated
rate of diabetic retinopathy (p=0.04), when compared to diabetics
with the Leu7/Leu7-genotype.
[0043] Discussion
[0044] Our findings based on elderly Finnish non-diabetic and
diabetic subjects indicates that the Pro7 allele of preproNPY is
strongly associated with increased carotid atherosclerosis, and
even more markedly in diabetic patients. This finding is of
importance, because an increase in the thickness of IMT of carotid
arteries increases the risk for cardiovascular events in a linear
fashion even before clinical manifestations of cardiovascular
diseases (26). In addition, the presence of Pro7 polymorphism in
the preproNPY was significantly associarted with the rate of
diabetic retinopathy. The Pro7 allele was also associated with high
serum LDL cholesterol levels and apolipoprotein B-levels in lean
non-diabetic subjects (15), but this was not found in diabetic
patients regardless of their body weight.
[0045] Type 2 diabetes is a state characterized by markedly
increased risk of atherosclerosis and although known risk factors
contribute largely to the occurrence of diabetic macrovascular
diseases (27), a large proportion of this vascular burden remains
unexplained and search for other potential environmental, metabolic
and genetic contributors are warranted. In this study we show for
the first time that diabetic patients with Pro7 allele have higher
carotid IMT than those with Leu7/Leu7-genotype. Although this
finding was based on a limited number of subjects, the lack of
association of Pro7 allele with other risk factors measured in
diabetic patients makes the finding more intriguing. As
non-diabetic control group included subjects with impaired glucose
tolerance as any population-based study does and therefore, glucose
tolerance is in a way continuum in this study population, we
combined the groups in order to increase the statistical power of
the study for the analysis of covariance. In this analysis age,
diabetes, systolic blood pressure and clinical macrovascular
disease were, as previously reported (21), powerful explanatory
variables of carotid IMT. Interestingly, the effect of NPY genotype
remained statistically significant in this analysis. Other
cardiovascular risk factors except fasting insulin in non-diabetic
subjects were not associated with NPY genotype in either group. The
selective mortality may cause bias in the interpretation, as in any
cross-sectional analysis. However, as LDL-cholesterol-levels were
constantly higher during the whole 10-year follow-up in lean
non-diabetic control subjects with Pro7 allele and, on the other
hand, the genotype effect on carotid IMT was more marked in
diabetic patients who had high cardiovascular mortality from the
time of diagnosis (18), it is likely that this study
under-estimates this association.
[0046] Why could then NPY enhance the development of
atherosclerosis? First, this effect may be mediated by the effects
of the PreproNPY genotype on LDL-cholesterol metabolism (15).
However, this effect is modulated by body weight (15) and as judged
from the present study, no effect was seen in Type 2 diabetic
patients in this regard (more detailed arialysis of lipoproteins
assessed either cross-sectionally or longitudinally gave no further
insights in this regard). Second, NPY may have angiogenic
properties that could be implicated in the development of
atherosclerosis. NPY has been shown to act as a smooth muscle
mitogen (28), to stimulate attachment, migration, DNA synthesis
(29), and the formation of capillary tubes by human endothelial
cells (4). Minor proportion of circulating NPY level is derived
from endothelial cells and this endothelially derived NPY may act
as an autocrine angiogenic factor even at very low concentrations
(4). Subjects with Pro7 substitution in preproNPY may therefore be
predisposed to increased arterial wall thickening seen as increased
intima-media thickening of carotid arteries, because of impaired
function of endothelial NPY. Third, NPY is an important modulator
of autonomic nervous system. Majority of circulating NPY is derived
from the perivascular sympathetic nerve endings, and the level of
NPY is correlated to those of norepinephrine (30). Autonomic
nervous dysfunction is an independent predictor of cardiovascular
mortality in patients with Type 2 diabetes, as demonstrated from
this study population (22). The mechanisms behind cardiovascular
diseases and autonomic nervous dysfunction are speculative, but our
unpublished observations suggest that cardiac autonomic regulation
is altered in subjects with those with Pro7 substitution in the
preproNPY. Therefore, we suggest that atherosclerosis may be
associated with gene(s) involved in vascular development, lipid
metabolism and autonomic nervous function and the recently found
gene variant (15) in NPY is the first one in this respect shown to
be related to accelerated atherosclerosis.
[0047] In conclusion, these results indicate that the presence of
Pro7 substitution in the preproNPY is associated with
ultrasonographically assessed carotid atherosclerosis in Finnish
diabetic and non-diabetic subjects. Furthermore, this study
provides first evidence that the Pro7 in the preproNPY is also
associated with increased rate of diabetic retinopathy in NIDDM
(Type 2 diabetes) patients, which could be potential target for
drug development.
[0048] It will be appreciated that the methods of the present
invention can be incorporated in the form of a variety of
embodiments, only a few of which are disclosed herein. It will be
apparent for the specialist in the field that other embodiments
exist and do not depart from the spirit of the invention. Thus, the
described embodiments are illustrative and should not be construed
as restrictive.
1TABLE 1a The clinical characteristics of the study population
according to Leu7/Pro-genotype in nondiabetic subjects. Leu7/Leu7
Pro7/- Characteristic n = 90 N = 15 p-value Age (years) 65.5 .+-.
0.6 65.5 .+-. 1.1 0.982 Male gender (n, %) 43 (48) 5 (33) 0.298
Body mass index (kg/m.sup.2) 27.8 .+-. 0.5 28.4 .+-. 1.2 0.611
Macrovascular disease (n, %) 11 (12) 4 (27) 0.139 Smoking history
(n, %) 23 (26) 5 (33) 0.528 Treatment for hypertension 26 (29) 6
(40) 0.387 (n, percentage) Systolic blood pressure (mmHg) 149 .+-.
2 148 .+-. 3 0.863 Diastolic blood pressure (mmHg) 85 .+-. 1.1 85
.+-. 3 0.847 Fasting serum insulin (mU/L) 11.0 .+-. 0.6 14.8 .+-.
2.2 0.056 Impaired gluclose tolerance (n, %) 11 (12) 1 (7) 0.531
Mean of common carotid IMT 1.04 .+-. 0.2 1.14 .+-. 0.4 0.156 (mm)
Serum apolipoprotein B (mg/L) 1.04 .+-. 0.03 1.12 .+-. 0.08 0.285
Serum HDL cholesterol (mmol/L) 1.34 .+-. 0.03 1.27 .+-. 0.08 0.403
Serum LDL cholesterol (mmol/L) 4.11 .+-. 0.09 4.61 .+-. 0.30 0.05
Serum total cholesterol (mmol/L) 6.29 .+-. 0.11 6.72 .+-. 0.37
0.153 Serum triglycerides (mmol/L) 1.81 .+-. 0.12 1.65 .+-. 0.18
0.811 Waste-to-hip ratio 0.91 .+-. 0.01 0.91 .+-. 0.03 0.926
[0049]
2TABLE 1B The clinical characteristics of the study population
according to Leu7/Pro-genotype in diabetic subjects. Leu7/Leu7
Pro7/- Characteristic n = 73 n = 8 p-value Age (years) 67.1 .+-.
0.7 66.5 .+-. 1.2 0.765 Male gender (n, %) 36 (49) 5 (63) 0.479
Body mass index (kg/m.sup.2) 29.4 .+-. 0.6 27.6 .+-. 4.1 0.344
Macrovascular disease (n, %) 28 (38) 5 (63) 0.187 Smoking history
(n, %) 25 (34) 2 (25) 0.598 Treatment for hypertension 40 (55) 5
(63) 0.677 (n, percentage) Systolic blood-pressure (mmHg) 154 .+-.
2.8 150 .+-. 10.3 0.637 Diastolic blood pressure (mmHg) 84 .+-. 2
87 .+-. 4 0.482 Fasting serum insulin (mU/L) 15.0 .+-. 0.9 15.7
.+-. 3.7 0.823 Mean of common carotid IMT 1.18 .+-. 0.03 1.58 .+-.
0.21 0.004 (mm) Serum apolipoprotein B (mg/L) 1.17 .+-. 0.03 1.00
.+-. 0.09 0.10 Serum HDL cholesterol (mmol/L) 1.11 .+-. 0.03 1.27
.+-. 0.12 0.142 Serum LDL cholesterol (mmol/L) 4.09 .+-. 0.10 3.66
.+-. 0.27 0.204 Serum total cholesterol (mmol/L) 6.44 .+-. 0.16
5.95 .+-. 0.36 0.325 Serum triglycerides (mmol/L) 2.62 .+-. 0.22
2.09 .+-. 0.34 0.455 Waste-to-hip ratio 0.94 .+-. 0.01 0.98 .+-.
0.03 0.222
[0050]
3TABLE 2 Analysis of covariance for mean carotid
intima-media-thickness adjusting for the effectcs of Leu7/Pro
polymorphism and covariates in the combined cohort. Risk Factor
F-value Significance Age 7.744 0.006 Gender 2.866 0.092 Diabetes
3.960 0.048 NPY Leu7/Pro 5.165 0.024 Macrovascular disease 4.278
0.040 Smoking history 2.225 0.138 Systolic blood pressure 5.754
0.018 LDL-cholesterol 0.142 0.707 2-way interaction: diabetes X NPY
Leu7/Pro F = 0.174, p = 0.677
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Sequence CWU 1
1
9 1 325 DNA Homo sapiens 1 ccgcttcttc aggcagtgcc tggggcggga
gggttggggt gtgggtggct ccctaagtcg 60 acactcgtgc ggctgcggtt
ccagccccct ccccccgcca ctcaggggcg ggaagtggcg 120 ggtgggagtc
acccaagcgt gactgcccga ggcccctcct gccgcggcga ggaagctcca 180
taaaagccct gtcgcgaccc gctctctgca ccccatccgc tggctctcac ccctcggaga
240 cgctcgcccg acagcatagt acttgccgcc cagccacgcc cgcgcgccag
ccaccgtgag 300 tgctacgacc cgtctgtcta ggggt 325 2 247 DNA Homo
sapiens 2 cccgtccgtt gagccttctg tgcctgcaga tgctaggtaa caagcgactg
gggctgtccg 60 gactgaccct cgccctgtcc ctgctcgtgt gcctgggtgc
gctggccgag gcgtacccct 120 ccaagccgga caacccgggc gaggacgcac
cagcggagga catggccaga tactactcag 180 cgctgcgaca ctacatcaac
ctcatcacca ggcagaggtg ggtgggaccg cgggaccgat 240 tccggga 247 3 142
DNA Homo sapiens 3 acttgcttta aaagactttt ttttttccag atatggaaaa
cgatctagcc cagagacact 60 gatttcagac ctcttgatga gagaaagcac
agaaaatgtt cccagaactc ggtatgacaa 120 ggcttgtgat ggggacattg tt 142 4
300 DNA Homo sapiens 4 ccttacatgc tttgcttctt atgttttaca ggcttgaaga
ccctgcaatg tggtgatggg 60 aaatgagact tgctctctgg ccttttccta
ttttcagccc atatttcatc gtgtaaaacg 120 agaatccacc catcctacca
atgcatgcag ccactgtgct gaattctgca atgttttcct 180 ttgtcatcat
tgtatatatg tgtgtttaaa taaagtatca tgcattcaaa agtgtatcct 240
cctcaatgaa aaatctatta caatagtgag gattattttc gttaaactta ttattaacaa
300 5 551 DNA Homo sapiens CDS (87)..(377) sig_peptide (87)..(170)
5 accccatccg ctggctctca cccctcggag acgctcgccc gacagcatag tacttgccgc
60 ccagccacgc ccgcgcgcca gccacc atg cta ggt aac aag cga ctg ggg ctg
113 Met Leu Gly Asn Lys Arg Leu Gly Leu 1 5 tcc gga ctg acc ctc gcc
ctg tcc ctg ctc gtg tgc ctg ggt gcg ctg 161 Ser Gly Leu Thr Leu Ala
Leu Ser Leu Leu Val Cys Leu Gly Ala Leu 10 15 20 25 gcc gag gcg tac
ccc tcc aag ccg gac aac ccg ggc gag gac gca cca 209 Ala Glu Ala Tyr
Pro Ser Lys Pro Asp Asn Pro Gly Glu Asp Ala Pro 30 35 40 gcg gag
gac atg gcc aga tac tac tcg gcg ctg cga cac tac atc aac 257 Ala Glu
Asp Met Ala Arg Tyr Tyr Ser Ala Leu Arg His Tyr Ile Asn 45 50 55
ctc atc acc agg cag aga tat gga aaa cga tcc agc cca gag aca ctg 305
Leu Ile Thr Arg Gln Arg Tyr Gly Lys Arg Ser Ser Pro Glu Thr Leu 60
65 70 att tca gac ctc ttg atg aga gaa agc aca gaa aat gtt ccc aga
act 353 Ile Ser Asp Leu Leu Met Arg Glu Ser Thr Glu Asn Val Pro Arg
Thr 75 80 85 cgg ctt gaa gac cct gca atg tgg tgatgggaaa tgagacttgc
tctctggcct 407 Arg Leu Glu Asp Pro Ala Met Trp 90 95 tttcctattt
tcagcccata tttcatcgtg taaaacgaga atccacccat cctaccaatg 467
catgcagcca ctgtgctgaa ttctgcaatg ttttcctttg tcatcattgt atatatgtgt
527 gtttaaataa agtatcatgc attc 551 6 97 PRT Homo sapiens 6 Met Leu
Gly Asn Lys Arg Leu Gly Leu Ser Gly Leu Thr Leu Ala Leu 1 5 10 15
Ser Leu Leu Val Cys Leu Gly Ala Leu Ala Glu Ala Tyr Pro Ser Lys 20
25 30 Pro Asp Asn Pro Gly Glu Asp Ala Pro Ala Glu Asp Met Ala Arg
Tyr 35 40 45 Tyr Ser Ala Leu Arg His Tyr Ile Asn Leu Ile Thr Arg
Gln Arg Tyr 50 55 60 Gly Lys Arg Ser Ser Pro Glu Thr Leu Ile Ser
Asp Leu Leu Met Arg 65 70 75 80 Glu Ser Thr Glu Asn Val Pro Arg Thr
Arg Leu Glu Asp Pro Ala Met 85 90 95 Trp 7 12 RNA Homo sapiens
misc_feature (10) n is a u in the wildtype and c in the mutant 7
acaagcgacn gg 12 8 9 DNA Homo sapiens misc_feature (5) n is a t in
the wildtype and c in the mutant 8 cgacngggg 9 9 12 DNA Homo
sapiens 9 acaagcgacc gg 12
* * * * *