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A Functional Neuropeptide Y Leu7Pro Polymorphism Associated With Alcohol Dependence in a Large Population Sample From the United States
Jaakko Lappalainen, MD, PhD;
Henry R. Kranzler, MD;
Robert Malison, MD;
Lawrence H. Price, MD;
Christopher Van Dyck, MD;
Robert A. Rosenheck, MD;
Joyce Cramer, BS;
Steven Southwick, MD;
Dennis Charney, MD;
John Krystal, MD;
Joel Gelernter, MD
Arch Gen Psychiatry. 2002;59:825-831.
ABSTRACT
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Background Quantitative trait locus studies, and observations in animals manipulated
for the neuropeptide Y (NPY) gene suggest that variation within this gene
may contribute to alcoholism. A recent population study suggested that the Pro7 allele of a functional NPY polymorphism (Leu7Pro)
may be associated with increased alcohol consumption. We tested whether the Pro7 allele is associated with alcohol dependence in European Americans
(EA).
Methods The design was a population study comparing the Leu7Pro
allele frequencies in alcohol-dependent subjects and controls. Population
stratification potential and diagnostic specificity was studied by genotyping
individuals from additional populations and psychiatric diagnostic classes.
We studied 2 independently collected samples of EA alcohol-dependent subjects
(sample 1, n = 307; sample 2, n = 160) and a sample of psychiatrically screened
EA controls (n = 202); 8 population samples, including African Americans and
European Americans (total n = 551); and 4 samples of individuals with Alzheimer
disease, schizophrenia, posttraumatic stress disorder, and major depression
(total n = 502). The main outcome measure was the difference in Leu7Pro allele frequencies between alcohol-dependent subjects and controls.
Results The frequency of the Pro7 allele was higher in the alcohol-dependent
subjects (sample 1, 5.5%; sample 2, 5.0%) compared with the screened EA controls
(2.0%) (sample 1 vs controls, P = .006; sample 2 vs controls, P = .03).
The attributable fraction (excess morbidity) in similarly
affected populations, owing to the Pro7 allele, was estimated
to be 7.3%. The frequency of the Pro7 allele was equal or lower
in the population samples, as compared with the screened EA controls (0%-2.2%),
with 1 exception (Bedouins). We found no significant evidence that the association
of the Pro7 allele with alcohol dependence was due to an association
with a comorbid psychiatric disorder.
Conclusions These results suggest that the NPY Pro7 allele is a risk
factor for alcohol dependence. This is only the second specific genetic mechanism
ever identified that modulates risk for alcohol dependence.
INTRODUCTION
ALCOHOLISM IS a genetically influenced disorder. The largest twin studies
have established an estimated heritability of 50% to 60% for alcoholism,1-2 which is comparable to, or higher than,
the heritability estimates for some other common disorders of major public
health concern, such as non–insulin-dependent diabetes mellitus3 and cancer.4 The only
genes unambiguously associated with risk for developing alcoholism are certain
genes coding for alcohol metabolizing enzymes, such as alcohol dehydrogenase
and acetaldehyde dehydrogenase.5 However, variation
within these genes accounts for only a small proportion of the risk for individuals
of European or African ancestry, who constitute the great majority of the
US population.
Neuropeptide Y (NPY) is a 36–amino acid peptide neurotransmitter.
Animal studies have suggested that NPY is involved in the regulation of appetite,6 reward,7 anxiety,8 and energy balance.9
Recently, quantitative trait locus (QTL) linkage mapping studies and behavioral
observations of mice manipulated for the NPY gene have suggested that genetic
variation at this locus may contribute to heritability in animal models of
alcoholism. Thiele et al10 showed that mice
deleted for the NPY gene prefer drinking alcohol-water solutions vs water.
These mice were also less sensitive to the hypnotic and sedative effects of
alcohol than nondeleted mice. In contrast, mice that were manipulated to overexpress
NPY had a low preference for alcohol and were more sensitive to the sedative
and hypnotic effects of ethanol. Quantitative trait locus mapping studies
in alcohol-preferring and nonpreferring rats obtained the highest logarithm-of-odds
(lod) score to rat chromosome 4. Marker D4Mit7, which
is located in an intron of the NPY gene, was in the center of the identified 12.5cM critical region.11-12
In humans, a functional Leu7Pro polymorphism
in the NPY gene has been described.13 This
polymorphism resides in the signal peptide part of the pre-proNPY and has
been demonstrated to affect intracellular processing of the pre-proNPY and
release of the mature NPY. For example, individuals with the Pro7/Leu7 genotype have an average of 42% higher maximal increases
in the plasma concentration of NPY in response to maximal physiological stress
as compared with Leu7/Leu7 individuals.14
Large population studies conducted in Northern Europe, particularly in Finland,
have associated the Pro7 allele with higher cholesterol
levels13 and atherosclerosis.15
Consistent with the studies in animals, a recent large population study suggested
that this polymorphism might also influence alcohol consumption in the normal
range. In that study, Kauhanen et al16 reported
that the Pro7 allele is associated with an average
of 34% higher alcohol consumption in a cohort of 889 Finnish middle-aged men.
Thus, a possible relationship between genetic variation at the NPY locus
and alcohol dependence is supported by studies of genetically manipulated
animals,10 by animal QTL linkage studies,11-12 by demonstration of functional concomitants
of genetic variation at the locus,13-15
and by a relationship with alcohol consumption in a Finnish population,16 although not necessarily in a pathological range
in that study.
In this study, we tested whether the NPY Pro7
allele is associated with a greater risk of developing DSM-III-R or DSM-IV alcohol dependence in
a large population sample from the United States. Two independent samples
of European American alcohol-dependent subjects and a sample of psychiatrically
screened European American controls were studied. Potential for population
stratification was evaluated by determining the Leu7Pro allele frequencies in 8 additional population samples, including European
Americans, European Ashkenazi Jews, and African Americans, and by determining
the allele frequencies of the FY(+/-) polymorphism
in our control sample. The FY(-) allele denotes
a promoter polymorphism in the FY gene, which abrogates
the expression of Duffy receptor on erythroid cells. The frequency of the FY(-) allele in modern day African Americans is 80%
and only 0.9% in European Americans.17 Owing
to this difference, the frequency of FY alleles may
be used to evaluate the degree of admixture between these 2 major US ethnic
groups. Diagnostic specificity was tested by determining Leu7Pro allele frequencies in European American individuals with schizophrenia,
affective disorder, posttraumatic stress disorder (PTSD), and Alzheimer disease.
SUBJECTS AND METHODS
PATIENTS AND SAMPLES
All subjects in each group described here gave informed consent as approved
by the relevant institutional review board.
Alcohol-Dependent Subjects
Two independent populations of alcoholics were studied. The initial
sample was recruited from a combination of treatment-seeking and non–treatment-seeking
patient populations through clinics and advertisements in the community. All
subjects in the initial alcohol-dependent sample (n = 307, termed hereafter
"EA alcohol-dependent sample 1") were recruited in Connecticut, at either
the University of Connecticut Health Center (Farmington, Conn), or the Veterans
Administration (VA) Connecticut Healthcare System, West Haven campus. All
alcohol-dependent subjects were diagnosed using the DSM-III-R18 or DSM-IV.19 The diagnosis was made with the Structured Clinical
Interview for DSM-III-R or for DSM-IV (SCID),20-21 the
computerized Diagnostic Interview Schedule for DSM-III-R (C-DIS-R)22 or a checklist composed
of DSM-III-R symptoms. The second alcohol-dependent
sample (n = 160, termed hereafter "EA alcohol-dependent sample 2") was obtained
through a multicenter VA study on the efficacy of naltrexone in the treatment
of alcohol dependence (VA Cooperative Study 425, "Naltrexone in the Treatment
of Alcoholism"). Subjects were recruited at 15 VA hospitals nationwide. The DSM-IV diagnosis was made using the SCID. All subjects
in these 2 study populations were European Americans diagnosed with alcohol
dependence. Individuals with psychotic disorders were excluded. In all analyses,
these 2 alcohol-dependent groups were tested separately, and no pooled data
are presented.
Screened Control Subjects
Subjects were recruited from the local communities through advertisement.
All subjects were screened to exclude major Axis I disorders, including substance
use disorders (such as alcohol dependence), psychotic disorders, anxiety disorders,
and mood disorders. A total of 267 screened controls were collected at the
VA Connecticut Healthcare System, West Haven Campus, and at the University
of Connecticut Health Center. Screening was done using the SCID, the Schedule
for Affective Disorders and Schizophrenia—Lifetime Version23
(SADS-L), the C-DIS-R, or via a nonstructured interview with a psychiatrist
or research assistant. There were both European American (n = 202) and African
American (n = 65) screened control subjects. The screened African American
controls were used as a population sample. For clarity, the screened European
American control subjects will be termed the "screened EA controls" hereafter,
and the screened African American controls will be termed "African American
sample." Twenty-two percent of the screened EA controls were assessed using
an unstructured interview.
Population Samples
DNA from unrelated Ashkenazi Jews (n = 90), Ethiopian Jews (n = 47),
Bedouins (n = 32), Moroccans (n = 92), and Druze (n = 92) was obtained from
the National Laboratory for the Genetics of Israeli Populations, Sackler Faculty
of Medicine, Tel Aviv University, Tel Aviv, Israel. Samples from unrelated
Japanese subjects (n = 66) were provided by Hiroshi Ichinose, PhD, and Toshiharu
Nagatsu, PhD, at Fujita Health University, Toyoake, Japan. A sample of unrelated
elderly European American control subjects (n = 67) (without Alzheimer disease
diagnosis) was recruited at the Alzheimer's Disease Research Unit at the Yale
University School of Medicine, New Haven, Conn.
Alzheimer Disease Subjects
The study population was recruited at the Alzheimer's disease Research
Unit at the Yale University School of Medicine, and consisted of patients
with probable Alzheimer disease (n = 137), which was diagnosed according to
standard criteria.24 Other causes of dementia
were excluded through a comprehensive evaluation, including medical history,
physical and neurological examinations, extensive serum chemistry evaluations,
and brain computed tomography scans or magnetic resonance imaging scans. All
subjects with Alzheimer disease were European American.
PTSD Subjects
All subjects (n = 77) with PTSD were Vietnam-era combat veterans who
were recruited at the VA Connecticut Healthcare System, West Haven. Consensus
diagnoses were made with SCID. Individuals with major psychotic disorders
were excluded. All subjects with PTSD were European American.
Major Depression
Patients (n = 122) were recruited at either the Connecticut Mental Health
Center or the VA Connecticut Healthcare System, West Haven Campus. The diagnosis
of DSM-III-R major depression, past or current, single
episode or recurrent, was established by SCID or by consensus of 2 psychiatrists.
Individuals with major psychotic disorders were excluded. All subjects with
major depression were European American.
Schizophrenia
Patients (n = 166) were recruited at 12 VA hospitals nationwide through
VA Cooperative Study 17 comparing the efficacy and cost-effectiveness of haloperidol
and clozapine, or through a separate research protocol at the VA Connecticut
Healthcare System, West Haven Campus. The diagnoses were made with SCID. All
subjects with schizophrenia were European American.
GENOTYPING
DNA was extracted from whole blood using standard methods, or from cell
lines in the case of the samples obtained from Israel. The NPY Leu7Pro polymorphism was genotyped as previously described.13 Briefly, genomic DNA was amplified using standard
polymerase chain reaction (PCR) techniques. The PCR product was digested with
10 Units of BsiE1 enzyme (New England Biolabs, Beverly,
Mass) and analyzed on a 3% Metaphor gel (BioWhittaker Molecular Applications,
Rockland, Me). Randomly chosen samples (approximately 8% of the total) were
reamplified, digested, and reanalyzed for quality control purposes with 100%
confirmation of previously assigned genotypes. Among the 1722 subjects genotyped
for the Leu7Pro polymorphism in this study, only
one Pro7/Pro7 homozygote was found. That individual
was diagnosed with PTSD and alcohol dependence. The FY
polymorphism was genotyped as described previously.17
Briefly, genomic DNA was amplified using standard PCR techniques. The PCR
product was digested with 10 Units of RsaI enzyme
and analyzed on a 3% Metaphor gel.
STATISTICAL ANALYSIS
Contingency tables were used to compare allele frequencies between groups.
In the first set of analyses, EA alcohol-dependent sample 1 was compared with
the screened EA controls. This was followed by a comparison of EA alcohol-dependent
sample 2 with the screened EA controls using a similar analysis (Table 1). In the second set of analyses,
potential for population stratification was tested using heterogeneity analysis
of all population samples (Table 2).
In the third set of analyses, diagnostic specificity and potential for confounding
by an association to a comorbid disorder was tested using heterogeneity analysis
of the neuropsychiatric disorders (subjects with PTSD, major depressive disorder,
schizophrenia, Alzheimer disease, and EA alcohol-dependent sample 1) and screened
EA controls (Table 3). We performed
2 x 2 post hoc analyses to compare subjects with PTSD, major depressive
disorder, schizophrenia, and Alzheimer disease with the screened EA controls
(Table 3). Comparison of the EA
alcohol-dependent samples with the screened EA controls was done using 2 x
2 tables. Larger contingency tables were used for heterogeneity analyses of
both the population samples (2 x 9) and the set of samples with psychiatric
disorders (2 x 6). Heterogeneity analysis tested whether the samples
(cells) were drawn from a uniform distribution. In all cases, calculations
were done using the  2 test. We used the CLUMP program,25 which uses Monte Carlo simulation to estimate the
significance of the 2 statistic. In each analysis, 10 000
to 100 000 simulations were performed to obtain P
values for the 2 statistic. Results of the CLUMP T1 statistic
are presented. Genotype frequencies were tested for Hardy-Weinberg equilibrium
(HWE) using the HWSim program, which uses Monte Carlo simulation to test for
departure from HWE.26 Genotypes in all populations
and for all psychiatric disorders were in HWE. Attributable fraction (also
referred to as etiologic fraction) was calculated using the formula Fc(R-1)/R,
where Fc is the fraction of cases with the risk factor and R is the incidence-density
ratio (when using incidence cases) or prevalence odds ratio (when using prevalent
cases).27 The calculations for the attributable
fraction were done using genotypes, counting the presence of the Pro7 allele as a risk factor. Power analysis for transmission disequilibrium
test was calculated using the method described by Risch and Merikangas28 (implementation available at: http://www.mds.qmw.ac.uk/statgen/dcurtis/software.html).
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Table 1. Frequency of NPY Leu7Pro Alleles
in 2 Independent Populations of EA Alcohol-Dependent Subjects and Screened
EA Controls*
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Table 2. Frequency of the NPY Leu7Pro Alleles in the Population Samples,
EA Alcohol Dependent Sample 1 and Screened EA Controls*
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Table 3. Frequency of the NPY Leu7Pro Alleles Among Alcohol-Dependent
Subjects and Healthy Controls in Relation to the Subjects With Psychiatric
Disorders*
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RESULTS
COMPARISON OF NPY Leu7Pro ALLELE FREQUENCIES
IN ALCOHOL-DEPENDENT SUBJECTS AND SCREENED EA CONTROLS
The frequency of the Pro7 allele was significantly
higher in EA alcohol-dependent sample 1 than in the screened EA controls (21 = 7.80, P = .006). The frequency
of the Pro7 allele was also significantly higher
in EA alcohol-dependent sample 2 as compared with the screened EA controls
(21 = 5.08, P = .03).
These results are presented in Table 1.
COMPARISON OF NPY Leu7Pro ALLELE FREQUENCIES
IN POPULATION SAMPLES
Analysis of Leu7Pro allele frequencies in the
population samples (Bedouins, Moroccans, Ashkenazi Jews, elderly European
American controls, Ethiopian Jews, Druze, African Americans, and Japanese)
and screened EA controls revealed significant heterogeneity (28 = 21.2, P = .008). These results are presented
in Table 2.
COMPARISON OF NPY Leu7Pro ALLELE FREQUENCIES
IN PSYCHIATRIC DISORDERS
Analysis of Leu7Pro allele frequencies in psychiatric
disorders (EA alcohol-dependent sample 1, PTSD, and subjects with schizophrenia,
Alzheimer disease, and major depression) and screened EA controls revealed
suggestive heterogeneity (25 = 9.95, P = .08); 2 x 2 comparisons of subjects with PTSD, schizophrenia,
Alzheimer disease, and major depression with the screened EA controls were
not significant. These results are presented in Table 3.
FY(+/-) POLYMORPHISM IN THE SCREENED
EA CONTROL SUBJECTS AND IN THE AFRICAN AMERICAN SAMPLE
The FY(+/-) polymorphism was genotyped
in 75 screened EA control subjects (37% of individuals in the screened EA
control sample) and 41 individuals from the African American sample (63% of
individuals in the African American sample). All of the individuals belonging
to the screened EA control group were homozygous for the FY(+) allele. The frequencies of the FY(+)
and FY(-) alleles in the African American sample
were 0.20 and 0.80, respectively.
COMMENT
In this study, we tested whether the NPY Leu7Pro
polymorphism is associated with alcohol dependence. We derived the hypothesis
from previous findings in animals and humans suggesting that genetic variation
within the NPY locus may be associated with an altered risk to develop alcoholism-related
traits and behaviors.10-12,16
We discovered an increased Pro7 allele frequency
among EA alcohol-dependent subjects compared with screened EA controls. This
was observed in 2 independently collected samples of alcoholics. This suggests
that the Pro7 allele is associated with alcohol dependence.
A case-control strategy is vulnerable to confounding by population stratification,
and we sought to address this by genotyping the NPY Leu7Pro in additional population samples, including African Americans, Ashkenazi
Jews, and in a second sample of European Americans (elderly EA controls) collected
in the same geographical region as the first study sample. Confounding by
population stratification refers to a situation in which a noncausal association
is observed because of differences in allele frequency for the marker of interest
that occur based on population differences alone. Such differences by population
are observed for most genetic markers, and we report here that they are indeed
present for NPY Leu7Pro (Table 2). Thus, it is demonstrated to be important for case-control
studies of this NPY polymorphism to establish that the case and control samples
are well matched in terms of their respective population ancestries. Our population
observations allow us to identify certain specific kinds of stratification
(admixture) that could confound the results. In this study, except for Bedouins,
the Pro7 allele frequency was lower in all populations
studied as compared with the alcohol-dependent samples. Previously, Karvonen
et al13 have reported that the Pro7 frequency in Finns is approximately 6%. If these groups (Bedouins
or Finns) were greatly overrepresented in the alcohol-dependent groups, but
not in the control groups, the population difference (rather than a physiological
difference) could be driving the observed association. However, because the
individuals residing in the geographical region where EA alcohol-dependent
sample 1 and the screened EA controls were collected are not predominantly
of Finnish or Bedouin descent, we have no reason to believe that population
stratification explains the elevated Pro7 frequency
among alcohol-dependent individuals. Our study design does not fully eliminate
the possibility that population stratification is confounding the elevated Pro7 frequency in alcohol dependence; there could still
be admixture that is not readily observable (eg, between different Northern
European populations). Such admixture is much less likely to account for an
association like that reported here because European populations tend to have
similar marker allele frequencies. While there are no data to support the
assumption that NPY allele frequencies are similar in the full range of European
populations that might be ancestral for our sample, we do demonstrate that
3 different, and differently-ascertained, European populations (our screened
EA controls, elderly controls, and Ashkenazi Jews) have similar Pro7 allele frequency, and this markedly decreases the likelihood that
our positive result is accounted for by occult stratification within European
populations. Replication attempts to confirm the association between the Pro7 allele and alcohol dependence in US populations are
clearly needed, but it should be noted, however, that assuming similar Leu7Pro allele frequencies, a sample of at least 640 individuals
needs to be tested to gain a statistical power of 0.8 ( = .01). Studies
that use either the transmission disequilibrium test or a linkage approach
would be useful but relatively difficult to do, as it would be difficult to
ascertain sufficient numbers of families segregating the Pro7 allele, and because of loss of statistical power if flanking genetic
markers are used (since presumably, all "real" association information would
be derived from linkage disequilibrium with the functional Leu7Pro marker). We estimate that more than 200 parent-offspring trios
would be required to confirm this finding using transmission disequilibrium
testing (again, for power of 0.8; = .01).
As the frequency of the Pro7 allele was 0%
in the African American sample, it was remotely possible that the Pro7 frequency among the screened EA controls was deflated due to admixture
with the African American population. Several lines of evidence argue against
occurrence of this particular kind of admixture in our sample. First, as mentioned
earlier, in the 2 other European populations that we studied (Ashkenazi Jews
and elderly EA controls), the Pro7 allele frequency
was similar to the observed frequency in the screened EA controls. Second,
to further test for the presence of African American admixture in the screened
EA controls, we determined the frequency of the FY(-)
allele of the FY(+/-) polymorphism in part
of the screened EA control group. The frequency of the FY(-) allele in modern-day African Americans is 80% and only
0.9% in European Americans.17 We found that
the frequency of the FY(-) allele in the screened
EA control group was 0%, suggesting that no significant African American admixture
was present. It is unlikely that the low Pro7 allele
frequency in the screened EA controls is a result of admixture with other
populations, such as Asian Americans, since African Americans are the only
sizeable minority population in this geographical region.
We next examined the specificity of the elevated Pro7 frequency for alcohol dependence. Earlier reports that used plasma
and cerebrospinal fluid NPY levels and yohimbine-stimulated NPY release as
indices of NPY function implicated this neurotransmitter system in schizophrenia,29 depression,30 and
PTSD.31 All of these disorders are also highly
comorbid with alcohol dependence.32 It was
therefore possible that the association of the Pro7
allele to alcohol dependence was due to an association with a comorbid psychiatric
disorder. We tested whether the Leu7Pro polymorphism
is associated with schizophrenia, PTSD, major depression, and Alzheimer disease.
These populations were collected independently of the alcohol-dependent and
control subjects through separate research protocols focusing on recruiting
individuals suffering primarily from these disorders. As compared with the
alcohol-dependent samples, the absolute Pro7 allele
frequency was lower in each of the other psychiatric disorders studied, but
higher than the frequency in the screened EA controls. The heterogeneity analysis
that included all the subjects with disorders and screened EA controls revealed
a suggestive significance (P = .08). Post hoc 2 x
2 comparisons revealed that the Pro7 allele was not
significantly associated with psychiatric disorders other than alcohol dependence.
The sample sizes were smaller for the other disorders, and we cannot exclude
that a positive association would be observed in larger samples. It may be
concluded, however, that it is unlikely that the association between the Pro7 allele and alcohol dependence is driven by an association
of the Pro7 allele with any of the other disorders
we studied. The elevated Pro7 allele frequency showed
some specificity to alcohol dependence, but there was also a general trend
toward higher Pro7 frequency in the other disorders.
This was most notable in those with PTSD and schizophrenia. We hypothesize
that this may be driven by the high rate of comorbidity between alcohol dependence
and these psychiatric disorders. For example, in the PTSD sample, the prevalence
of alcohol dependence was higher than 50%, and the frequency of the Pro7 allele among these individuals (ie, subjects with
PTSD who had known comorbid alcohol dependence) was 5%. Another possibility
is that the genetic risk for these different psychiatric disorders is partially
shared. For example, one may speculate that the Pro7
allele is related to poor stress tolerance,33
which in some individuals may contribute to alcohol dependence, while in others
it may contribute to psychotic disorders or to a tendency to respond maladaptively
to traumatic events.
What is the significance of the Pro7 allele
in the development of alcohol dependence among European Americans? The attributable
fraction, which is the proportion of the cases in the study population that
would not have occurred had the risk factor (eg, a risk allele) not been present,
is a useful measure to quantify the impact of the Pro7
allele on the development of alcohol dependence.27
We used the recent census34 and the reported
14.1% DSM-III-R lifetime population prevalence of
alcohol dependence35 to estimate the attributable
fraction in European Americans who are 16 years of age or older. The attributable
fraction due to the Pro7 allele is 7.3%. Due to the
high prevalence of alcohol dependence in the United States, the estimated
number of cases that the Pro7 allele accounts for
among European Americans is higher than 1 800 000, assuming that
our sample is representative of all cases in the population.
How does altered NPY function affect the risk to develop alcohol dependence?
Kallio et al14 suggested that Pro7/Leu7 individuals have an enhanced capacity to synthesize and release
mature NPY. They showed that such individuals have an average of 42% higher
maximal increases in the plasma concentrations of NPY under maximal physiological
stress. Kallio et al14 also demonstrated that
human umbilical vein endothelial cells from Pro7/Leu7
individuals abundantly contain more mature NPY as compared with the human
umbilical vein endothelial cells from Leu7/Leu7 individuals.
Based on these findings and the findings from animal studies showing an inverse
correlation between NPY expression and alcohol preference, one would assume
that the Pro7/Leu7 individuals have a lower risk
of developing alcoholism. The present findings and those of Kauhanen et al16 suggest the opposite. It is possible, however, that
an exaggerated NPY release in Pro7/Leu7 individuals
may lead to a rapid depletion of NPY stores causing a prolonged nadir in the
baseline NPY levels. This mechanism was recently postulated by Morgan et al.33 They demonstrated that NPY is released in response
to stress (military survival training). Interestingly, a subpopulation of
their study subjects had significantly lower NPY plasma concentrations 24
hours after the stress, as compared with their prestress baseline. Other possible
intermediate phenotypes may involve differences in alcohol sensitivity36 and differences in alcohol's reward properties7 between the individuals with and without the Pro7 allele. More research is needed to understand the
pathways that connect the functional NPY Leu7Pro
polymorphism and the risk of developing alcoholism.
Our findings suggest that the NPY Pro7 allele
contributes to the heritability of alcohol dependence. If confirmed, this
would be only the second known specific mechanism by which genetic variation
leads to differing risk for alcohol dependence. Although independent replication
is needed, we note that our present finding was consistent across 2 differently
ascertained alcohol-dependent samples. Also, although we have no control sample
that can be strictly considered a replication sample, our results in elderly
controls and unselected Ashkenazi Jews (of European origin) suggest that the
low Pro7 allele frequency in our screened EA control
sample was not caused by a sampling error. Our findings are consistent with
recent studies in animals and humans, which indicate that genetic variation
within the NPY system may account for a proportion of the heritability of
volitional alcohol intake. It is interesting that the Pro7 allele seems to contribute to a spectrum of phenotypic variation,
ranging from increased average alcohol consumption16
to pathological drinking, as defined by DSM-III-R
or DSM-IV criteria of alcohol dependence. Our findings
are limited to European Americans, mostly due to the lack of access to alcohol-dependent
samples from other populations. The Pro7 allele was
absent in the African American or Japanese samples, suggesting that this polymorphism
is unlikely to play a significant role in the risk for alcohol dependence
in these populations. Studies aiming to elucidate the pathways that connect
the NPY Leu7Pro polymorphism and risk to develop
alcoholism will be important. These studies may shed light on some of the
neurobiological mechanisms and pathways that lead to the development of alcohol
dependence, and may thereby facilitate the development of novel treatments
and prevention strategies for this common and debilitating disorder.
AUTHOR INFORMATION
Submitted for publication October 10, 2001; final revision received
October 12, 2001; accepted October 12, 2001.
This work was supported by grants K02-MH01387, R01-AA11330, K02-AA00239,
P50-AA03510, R21-DA10242, K02-AA00261, P50-AA12870, M01-RR06192, and IK08AA
13732-D1 from the National Institutes of Health, Bethesda, Md (University
of Connecticut General Clinical Research Center) and the US Department of
Veterans Affairs, Washington, DC (the Alcohol Research Center, National Center
for PTSD, and the VA Connecticut-Massachusetts Mental Illness Research, Education
and Clinical Center).
Presented in part at the Annual Meeting of the American Congress of
Neuropsychopharmacology, December 11th 2000, San Juan, Puerto Rico.
We thank Ellen Hibbard, BS, Kathleen Bonvicini, MPH, and Ann Marie Lacobelle,
MS, for excellent technical support. We thank Hiroshi Ichinose, PhD, and Toshiharu
Nagatsu, PhD, at the Fujita Health University, Toyoake, Japan, for providing
the Japanese DNA samples. We thank Dan A. Oren, MD, for the depression samples
he contributed. We also thank all the collaborators in VA Cooperative Study
425 ("Naltrexone in the Treatment of Alcoholism") for providing us with the
samples for the EA alcohol-dependent sample 2, and the collaborators in VA
Cooperative Study 17 ("Clozapine versus Haloperidol for Schizophrenia") for
the EA schizophrenia sample.
Corresponding author and reprints: Joel Gelernter, MD, Yale University
School of Medicine, Department of Psychiatry, VA Connecticut Healthcare System,
950 Campbell Ave, Psychiatry 116A2, West Haven, CT 06516 (e-mail: joel.gelernter{at}yale.edu).
From the Department of Psychiatry, Yale University School of Medicine,
New Haven, Conn (Drs Lappalainen, Malison, Van Dyck, Rosenheck, Southwick,
Charney, Krystal, and Gelernter, and Ms Cramer), the Veterans Administration
Connecticut Healthcare System, West Haven (Drs Lappalainen, Rosenheck, Southwick,
Krystal, and Gelernter, and Ms Cramer), the Department of Psychiatry, University
of Connecticut School of Medicine, Farmington (Dr Kranzler); and Butler Hospital
and the Department of Psychiatry and Human Behavior, Brown University School
of Medicine, Providence, RI (Dr Price).
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