|ACE; NPY; Polymorphisms; Risk factor; Atherosclerosis;
Cardiovascular disease; Left ventricular hypertrophy
|A number of factors contribute to the etio-pathogenesis of
multifactorial diseases . The identification of the sequence of events
and of the acting molecules might help to define the natural history
of multifactorial diseases, to ameliorate the diagnostic approach and
to refine the prognosis, paving the way to novel molecular therapeutic
|Environmental and genetic factors were analyzed in order to
elucidate the nature and the role of the elements involved in the
individual liability to multifactorial diseases. A number of studies
reported associations of polymorphism with different pathological
conditions [1-3]. Many studies found significant associations, whereas
others did not confirm those findings [3,4]. Numerous factors, such
as the matching factors or other linked genetic markers, could play
confounding role upon results. Despite the controversial findings, great
interest raised around selected polymorphisms associated to a number
of multifactorial diseases, with special regard to atherosclerosis,
cardiovascular and metabolic diseases [2-4].
|We resumed literature data about polymorphisms of two genes,
which codify respectively for neuropeptide Y (NPY) and Angiotensin
converting enzyme (ACE), both molecules involved in the pathogenesis
of cardiovascular disease (CD) and atherosclerosis . We also present
a case report of a male, suddenly deceased from myocardial infarction,
presenting with left ventricular hypertrophy (LVH) at post-mortem
examination, otherwise clinically silent. In the presented case, we
analysed polymorphisms of NPY and ACE genes.
|Materials and Methods
|A 40 year old man suddenly had a collapse in a public store.
Resuscitation attempts were unsuccessful and he died in a few minutes.
Familial anamnestic data did not record cardiovascular or metabolic
diseases. The patient was referred in good health and no information
was available regarding his blood pressure.
|The body length was 185 cm, weight 100 kg, with a body mass index
(BMI) of 29.21. The patient was in good general hygienic conditions,
with normal muscle trophism and abundant android fatty tissue
distribution. Toxicological examination was negative for drug and
alcohol abuse. The heart weighed 970 g, with longitudinal diameter of
16cm, transverse diameter of 15 cm and antero-posterior diameter of
8.5 cm. Heart’s consistency was significantly increased. The section of
the heart apex showed important LVH: the thickness was was 4.0 cm,
4.1 cm in the septum and 3.3 cm at the apex level. The myocardium
was pale (boiled flesh-colour) with no macroscopic signs of necrosis.
The histology examination confirmed concentric hypertrophy. The
papillary muscles were significantly hypertrophic and increased in
consistency, and cordae tendineae were slightly thickened. The valvular
systems showed no alterations. The shape of the lungs was increased,
as well as the volume and consistency. The right lung weighed 820g
and the left 780g. The pulmonary tissue appeared smooth, moist, dark
red, presenting petechiae in the interlobar fissures. Large, medium and
small bronchi showed patent lumen and slightly hyperaemic mucosa.
Histology findings identified congestive aspect of the respiratory
mucosa. The histology of elastic arteries wall identified mild to moderate
atherosclerosis evidences, especially in the abdominal aorta wall.
|Molecular biology analyses
|The polymorphism analyses were conducted by Geneticlab srl (Noventa Vicentina, Italy). Briefly, RNA was extracted from frozen
heart tissue biopsy using TRIzol reagent (Invitrogen Corporation,
Carlsbad, CA, USA), spectrophotometrically quantified at 260 nm,
reverse-transcribed to cDNA using High Capacity cDNA Reverse
Transcription Kit (Applied Byosystems, Carlsbad, CA) and
amplified using GoTaq® qPCR Master Mix (Promega, WI, USA).
The following primer pairs (Bio Basic Inc., Amherst- NY, USA)
were used for real-time amplification of ACE gene insertion/deletion
allele: ACE1 primer 5′-CATCCTTTCTCCCATTTCTC-3′, ACE2
primer 5′-TGGGATTACAGGCGTGATACAG-3’, and ACE3
primer 5′-ATTTCAGAGCTGGAATAAAATT-3′. Amplification
and detection were performed using the CFX96™ Detection System
(Biorad Laboratories, Hercules, CA). For RT-PCR amplification
of the NPY 399T>C polymorphism, the following primers were
used: forward 5’- TTGCCTCACTCCAACAGCG-3’ and reverse 5’-
ACAACACCAAAGCCCAAGTATCT-3’. The amplification reaction
was conducted in Techne T-512 thermocycler (Techne, Staffordshire,
UK) and the sequencing of the amplification product was obtained
using the ABI PRISM 310 Genetic Analyzer (Applied Biosystems,
Carlsbad, CA). The PCR products were digested by BsiEI (New
England Biolabs, Beverly, Massachusetts) and digestions were analysed
by electrophoresis on 2% agarose gel. For quality control, we used
samples at random for repeat analysis using our standard genotyping
protocol, which were in 100% concordance with the original.
|ACE: genotype ID. Analysis of PCR products on 2% agarose gel
electrophoresis under UV light by staining etidium bromide detected
a band of 190 bp shows deletion (D) and a band of 490 bp shows
|NPY: genotype -399 T>C. We determined a T to C polymorphism
which results in a substitution of leucine to proline substitution at
codon 7 in the NPY gene. The percentage of efficiency was 100%.
|Selected polymorphisms of both ACE and NPY genes are
considered to be risk factors for CD [2,3], as well as for metabolic
diseases, including atherosclerosis [4-6]. However, the relationship and
the specific role of those polymorphisms are not fully clear.
|The post-mortem evaluation of our patient identified LVH, a
major risk factor underlying coronary heart disease, and molecular
biology analysis revealed the carrier status for selected ACE and NPY
polymorphisms, respectively ACE genotype I/D and NPY genotype
|NPY and CD
|The neuroendocrine regulation of the stress response is a complex
event, modulated and influenced by polymorphisms of stress-related
genes [6-11], including the NPY gene, which codifies for the most
abundant peptide in heart and brain [9-11]. NPY is produced by
sympathetic neurons, endothelial cells , and platelets . NPY
is involved in a number of functions including sympathetic nerve
stimulation, immune regulation , food intake control ,
coronary blood flow, ventricular function , vascular smooth muscle
cell proliferation and modulation of the contractile function during the
development of cardiomyocite hypertrophy [9-11]. NPY is associated
with pathological conditions, including schizophrenia , and eating
disorders . Measurement of NPY plasma levels contributes to
predict cardiovascular complications in end-stage renal disease affected patients [18,19]. Moreover, NPY is claimed to be involved in CD 
and congestive heart failure [20-22].
|The regulatory roles of NPY are largely determined by the
enzymatic processing of NPY (NPY1-36) and by its receptor subtypes
(NPY-Y1 to Y5). The regulation of the cardiovascular system activities
and of blood pressure by vasoconstriction and vascular smooth muscle
cell proliferation is mediated by NPY-Y1 receptor and its selective
agonist NPY (NPY1-36) [12-14,23-26]. Ischemic angiogenesis is under
the control of one or both NPY-Y2 or NPY-Y5 receptor subtypes and
the N-terminally truncated form of NPY (NPY3-36) [12,27,28].
|The NPY gene (NPY; OMIM *162640) is located on the short
arm of chromosome 7 (7p15.1). Selected NPY polymorphisms are
associated to cortisol and ACTH response to acute psychosocial stress.
In Scandinavian populations, one NPY variant, otherwise rare in
other populations, was associated with hyperlipidaemia and carotid
atherosclerosis [20-22], coronary alterations in type 1 diabetes ,
and myocardial infarction following hypertensive status .
|Many studies investigating the relationship between NPY
polymorphisms and disease risk focused on the non-synonymous
L7P (rs16139) polymorphism in preproneuropeptide Y (preproNPY),
that might induce abnormal local NPY signalling . The L7P
polymorphism otherwise increases the risk for type 2 diabetes and CD
concomitantly with further risk factors, such as obesity and hypertension
[30,31]. Furthermore, Pro7 substitution for Leu7 is related to increased
serum total cholesterol and low density lipoprotein levels in obese
Caucasian individuals . The L7P polymorphism is rare in Asians
[29,32,33]. The association of ischemic stroke with polymorphisms
TA and CC, constructed by two further polymorphisms, rs16135 and
rs16476 was reported.
|The intrinsic polymorphism rs16141 of NPY was related to
cerebrospinal fluid cholesterol levels in Alzheimer disease (AD)
patients [25-38], was reported in schizophrenia , and non-Hodgkin
lymphoma . It also affects stress response and emotion [28,35,36].
|Linkage scans suggested that chromosome 7p might be involved
in thermogenesis [37-41], as well as in pulse pressure, a measure of
central arterial stiffness and a predictor of CD-related mortality
. In vascular homeostasis NPY plays two opposite roles, vasodilation
or constriction, depending on control of the nervous systems
. Probably NPY polymorphisms, especially those involving the
gene expression regulatory regions, may affect the vascular system.
Special attention was paid to another NPY promoter polymorphism,
C-399T, also known as rs16147 and C-485T [32-36]. A putative SP1
transcription factor binding site within the rs16147 stretch of NPY
sequence is lost with the rs16147 an allele . The rs16147 G allele
(reverse strand C allele) overall increases the expression of the NPY
gene [38-41] and subsequently the NPY plasma levels, thus promoting
arterial smooth muscle cell proliferation  and thermogenesis
[21,32]. C-399T was related to ischemic stroke with higher prevalence
of hypertension affected individuals. Allele-specific effects of the
C-399T polymorphism were reported [18,32,38,39]. Controversial
observations were published  and no conclusive observations were
reported. However, the effect of NPY on stroke susceptibility seems to
act independently from hypertension . The hypothesis that NPY
contributes to atherosclerosis is supported by both the correlation
of plasma NPY levels with selected NPY polymorphisms, and by the
reported inhibition of murine atherosclerosis with an antagonist of the
NPY1 receptor, which mediates most cardiovascular effects of NPY .
In the present case report, the patient carried the rs16147 or C-399T genotype. Therefore, the identified polymorphism might indicate the
liability to arterial smooth muscle proliferation and thermogenesis.
Histology findings confirmed a moderate arterial atherosclerosis.
Unfortunately, an attempt to measure the patient’s plasma levels of
NPY failed, probably due to the post-mortem interval.
|A deeper understanding of the relationship among NPY,
cardiovascular risk factors and clinically diagnosed CD will deserve
further studies. The analysis of NPY polymorphisms might help to
refine the risk estimates for selected individuals displaying a familial
high risk predisposition to CD and/or atherosclerosis. In fact,
individuals which share a genetic liability to their NPY risk profile
might represent a prime target for prevention.
|ACE polymorphisms, hypertension and atherosclerosis
|ACE contributes to the finely tuned long-term regulation of blood
pressure and volume . ACE enzyme, a zinc metallopeptidase
widely distributed on the surface of endothelial and epithelial cells,
plays a pivotal role in converting the inactive decapeptide, angiotensin
I (Ang I or Ang 1-10), to the active octapeptide angiotensin II (Ang
II or Ang 1-8), a powerful vasoconstrictor main active product of the
renin-angiotensin system [46-49]. Ang II induces the adrenal cortex to
release aldosterone , mediates cell growth and proliferation, ,
and contributes to endothelial dysfunction by reducing nitric oxide
bioavailability . ACE also plays a critical role in the kinin-kallikrein
cascade, which also contributes to regulate the blood pressure [53,54].
|The human ACE gene (OMIM +106180), located on the long arm
of chromosome 17 (17q23), is 21 kilo bases (kb) long and comprises 26
exons and 25 introns. More than 160 ACE gene polymorphisms were
described, most of which are single nucleotide polymorphisms (SNPs)
[55,56]. The gene codifies for two isoforms: the somatic form (sACE),
with a molecular mass of 170 kDa, expressed in somatic tissue, and
the testicular form (tACE, germinal ACE -gACE), with a molecular
mass of 100 kDa, exclusively expressed in male germinal cells . A
homologue of ACE, ACE2, was cloned from human heart failure and
lymphoma cDNA libraries [57,58]. ACE2 gene contains 18 exons and
maps to Xp22 . In vitro studies indicate that the catalytic efficiency
of ACE2 for Ang II is 400-fold greater than for Ang I .
|The potential role of ACE and ACE2 as cardioprotective peptides
with vasodilator, antigrowth, and antiproliferative actions was recently
|Inter-individual differences in plasmatic ACE levels were described,
suggesting efficient long-term tuning of plasma levels, possibly due
to genetic regulation . A polymorphism involving the presence
(insertion I) or absence (deletion, D) of a 287-bp sequence of DNA
in intron 16 of the gene (NCBI ref. SNP ID: rs1799752) was described
. Mean ACE activity levels in DD carriers were approximately
twice those found in II genotype individuals . Subjects with the
ID genotype had intermediate levels, indicating allele codominancy.
Involvement of the I/D polymorphism was detected in plasma, as well
as in tissue ACE levels [64,65].
|Beside the involvement in neurodegenerative diseases, such as
AD [66-69], or metabolic illnesses, such as diabetes [70,71], ACE
polymorphisms were investigated in a number of pathologies of the
cardiovascular system in different populations. However, the complexity
of association and linkage studies, as well as misclassifications of
phenotypes may have lead to false-negative or false-positive results,
and controversial observations were reported.
|Although the nature, the specific role and location of ACE polymorphisms remain to elucidate, the I/D polymorphism is actually
considered a valid marker to investigate the relationship between
genetic predisposition and diseases. In normotensive men, after
infusion of Ang I, venous levels of Ang II and increases in blood
pressure were higher in DD carriers compared with II carriers .
However, controversial observations were reported . Anomalies in
further ACE activities, such as degradation of bradykinin, were also
investigated . Studies reported a positive association between the D
allele and high blood pressure [75-85]. Significant relationship between
the D allele and hypertension in women and in Asians was reported
. Controversial results were also reported in experimental models
. The possible association between the ACE I/D polymorphism and
atherosclerosis was actively investigated using arterial intima-media
thickness (IMT) measurement, a promising marker for very early
metabolic disease liability, even in foetuses [86-89]. Positive association
between the D allele and common carotid IMT in adults was reported
demonstrating results concordance between Caucasians and Asians
. The association was stronger among high-risk individuals,
including subjects with underlying diseases such as cerebrovascular
disease, diabetes, or hypertension . The association between
ACE genotype and atherosclerosis was also assessed using coronary
calcification as a measure of coronary atherosclerosis [90,91], and postmortem
measurements of aortic atherosclerosis [92,93]. However, also
in this case, the results were controversial and inconclusive.
|The D allele, considered as relevant exclusively in selected groups
of patients, is associated to changes in the left ventricle [89-94]. The
DD genotype was identified more frequently in male patients with
myocardial infarction than in normal controls, particularly among
low-risk controls, intended as individuals bearing low body mass index
and low plasma levels of apolipoprotein B (ApoB) . However,
this observation was not confirmed in a large association study
[95,96]. Association between the ACE polymorphism and physical
performance was also described [97-103]. The association of the I allele
with improved endurance is in line with the association of the D allele
with LVH. Carriers of the D allele may develop hypertrophy following
lower metabolic efficiency . Moreover, a recently published
report argued that the frequent association between short telomeres
and cardiovascular risk factors and/or age related diseases is affected
by the ACE genotype in the elderly hypertension subjects with LVH
. Genetic and epidemiological data documented that elevated
plasma ACE levels increase LVH risk . Moreover, animal studies
suggested the contribution of cardiac ACE levels to atrial enlargement
and cardiac arrhythmia . Probably, genetic anomalies leading to
ACE overexpression play an essential role in the pathogenesis of LVH.
A significant association of ACE gene I/D polymorphisms with LVH,
especially in East Asians, was described, with higher risk in males .
In the present case report, the molecular biology analyses identified the
ID carrier status. However, histology findings did not identify strong
evidence of IMT, although evidences of moderate aortic atherosclerosis
were detected. Unfortunately, we do not have previous biochemical
data regarding the plasma levels of ApoB in the patient.
|Further studies are required in order to evaluate the role and the
meaning of the D allele presence in the regulation of blood pressure
and/or the increase in the left ventricle wall thickness. In fact, ACE
polymorphisms might offer a powerful tool to identify high risk
individuals in the population.
|Epidemiologic results report the association ACE or NPY
polymorphisms with atherosclerosis, metabolic, cardiovascular and/or related diseases. The identification and prognosis of patients at high risk
to develop CD and/or metabolic diseases might be greatly ameliorated
using genetic liability risk factors, such as the polymorphisms of ACE
and NPY genes. As an example, our patient was referred as a good health
young man. However, he presented clinically silent LVH and presented
ACE and NPY polymorphisms suggestive for CD/metabolic diseases
high risk. One might speculate that knowledge of carrying risk factors,
such as LVH and polymorphisms, might address to cardiological or
internal medicine evaluation and to modify the life style.
|Further investigations are required in order to investigate
the functional effects of polymorphisms with respect to the
molecular activities of ACE or NPY. To understand the meaning of
polymorphisms might clarify the etiopathogenesis of a number of
diseases, and also might help to improve the evaluation of patients,
offering a further prognostic tool, thus paving the way to personalize
molecular therapeutic strategies.
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