Current Drug Targets - CNS & Neurological Disorders, 2003,2,41-51
41
Candidate Gene Analysis of the Price Foundation Anorexia Nervosa
Affected Relative Pair Dataset
A.W. B e r g e n 1 , 2 t , M.Yeager1y2,R. Welch192, J.K. G a n j e i l , A. D e e p - S o b o s l a y l ~ 3 ,K. HaquelY2,
M.B.M. van den Bree1j4, D. Goldrnan195, W.H. B e r r e t t i n i 6 , W.H. Kaye1,7* and the Price
Foundation Collaborative Group (www.anbn.org)
lBiognosis U.S., Inc. (Dissolved), Gaithersburg, M D 20877, USA, 2Currently at the Core Genotyping Facility,
Advanced Technology Center, National Cancer Institute, Gaithersburg, M D 20877, USA, 3Currently at the
Clinical Brain Disorders Branch, NIMH, NIH, Bethesda, M D 20892, USA, 4Currently at the Division of
Psychological Medicine, University of Wales College of Medicine, Card$ United Kingdom, jLaboratory of
Neurogenetics, NIAAA, NIH, Rockville, M D 20852, USA, 6Center f o r Neurobiology and Behavior, University of
Pennsylvania, Philadelphia, PA 191 04, USA, 7Department of Psychiatry, University of Pittsburgh, Pittsburgh, PA
15213, USA
Abstract: The eating disorders are severe psychiatric illnesses with significant morbidity and mortality that
exhibit statistically significant familial risk and heritability, providing support for a molecular genetic
approach toward defining etiological factors. An emerging candidate gene literature has concentrated on
serotinergic and dopaminergic candidates. With the financial support of the Price Foundation, a group of
investigators initiated an international multi-center collaboration (Price Foundation Collaborative Group) in
1995 to study the genetics of anorexia and bulimia nervosa by collecting and analyzing phenotypes and
genotypes of individuals and their relatives affected with eating disorders. The first sample of families
collected by this collaborative group, known as the Price Foundation Anorexia Nervosa Affected Relative Pair
(AN-ARP) dataset, was ascertained on an proband affected with Anorexia Nervosa (AN), with relative pairs
affected with the eating disorders AN, Bulimia Nervosa or Eating Disorders Not Otherwise Specified [l].
Biognosis US.,Inc. was founded to identify and characterize candidate susceptibility genes for anorexia and
bulimia nervosa phenotypes in the Price Foundation eating disorder datasets. During 2000-200 1, Biognosis
US., Inc. developed and implemented a research program with a focus on the analysis of candidate genes
nominated by neurochemical characteristics of eating disorder patients [2], serotonergic and dopaminergic
candidate gene polymorphisms [3], neuroendocrine regulation of appetite [4], and by a positional hypothesis
from a linkage analysis of the AN-ARP dataset [5]. This report reviews the anorexia nervosa candidate gene
literature through 2001, the candidate gene research program implemented at Biognosis U S . , Inc. and selected
candidate gene findings in the AN-ARP dataset derived from that research program.
Key Words: Anorexia Nervosa, Candidate Gene, Indolamine, Catecholamine, Neuropeptide, Genotypes, Alleles, Haplotypes,
Transmission Disequilibrium Test.
INTRODUCTION
Anorexia Nervosa Phenotype
-
Anorexia nervosa (“AN’) affects 0.5% of women [6]
and carries a significant mortality risk [7]. Although the
majority of patients (70-80%) do recover, this may take
considerable time (5 - 6 years) [8]. The diagnostic criteria
for AN include: refusal to maintain body weight at or above
a minimally normal weight, fear of gaining weight even
though underweight, and a disturbance in the patient’s
perception of body weight or shape and its effect on selfevaluation, and amenorrhea [ 9 ] . Comorbidity of AN and
~
~~~~~
*Address correspondence to the author at the Department of Psychiatry,
381 1 O’Hara St, 600 Iroquois Building, University of Pittsburgh School of
Medicine, Pittsburgh, Pennsylvania, 15213, USA; Ph: (412) 647 9845; Email: kayewh@msx.upmc.edu
‘This manuscript does not represent the opinion of the NIH, DHHS or the
Federal Government.
1568-007WO3 $41.00+.00
other psychiatric disorders (e.g., depression, OCD, anxiety
disorders, bipolar disorder) and extremes of personality traits
have been described [ 10-141. The reported familial relative
risk is approximately 1 1 for AN [ 151, suggesting genetic
factors may play a role. This is confirmed by twin studies,
which have reported heritability rates ranging from 50 and
80% [16-201. These findings provide the basis for a
molecular genetic approach to the study of eating disorder
susceptibility factors.
-
Existing AN Candidate Gene Association Studies 2001
Published molecular genetic association studies on AN
through 2001 are reviewed below and in [21, 221. Candidate
gene association analyses to AN phenotype at sixteen genes
have been published in the English language literature as of
April 2001 (Table I and Table 11). These analyses compare
genotype and allele counts from one or more DNA
polymorphisms at candidate genes in individuals affected
0 2003 Bentham Science Publishers Ltd.
42
Current Drug Targets - CNS & Neurological Disorders 2003, VoI. 2, No. I
with AN (cases) versus individuals unaffected with AN
(controls), i.e., case:control contingency association
analysis. Case status was either DSM-IIIR or DSM-IV AN.
Typically, candidate gene case:control studies in eating
disorder samples have been based on a single polymorphism
at most of these genes, although two serotonin loci have
received attention from several groups and several groups
have screened a candidate gene for novel polymorphisms
(Table I). The mean AN sample size in these candidate gene
studies is approximately 90 individuals, while the mean
control sample is about 180 individuals. Control samples in
this candidate gene literature are very diverse, consisting of
both psychiatrically screened and unscreened samples,
normal weight, underweight and obese samples, and samples
of both sexes. Several groups of investigators have samples
that include parents or unaffected siblings. Family samples
permit both association analysis and linkage analysis and
reduce the probability of type I error (false positives) at the
cost of increased type I1 error (false negatives). Ideally, any
positive association findings in case:control samples must
be evaluated in additional case:control samples and in family
samples to increase confidence in a proposed relationship
between candidate gene sequence variation and susceptibility
to an eating disorder.
Serotonergic Candidate Gene Studies Through 2001
Bergen et at!
the rate limiting enzyme for serotonin synthesis in brain
(Table I). While substitution polymorphisms at the loci for
serotonin receptors 1B (Phel24Cys) and 7 (Pro-279-Leu)
have been previously associated with alcoholism [23] and
antisocial personality disorder [24], the result of association
studies between these candidate polymorphisms and AN was
non-significant [25]. A sequence polymorphism flanking the
HTR2A locus (-1438G>A) has been reported to be
significantly associated with both AN and OCD [26, 271,
and with OCD in females only [28]. However, some studies
of this HTR2A polymorphism have not exhibited
statistically significant association to AN, e.g., [29-3 I], and
see comments to [26]. A functional serotonin 2C receptor
locus substitution polymorphism (Cys23Ser) [32] has been
previously associated with hyperphagia and auditory
hallucinations in Alzheimer's disease [33]; but this
substitution was not found to be significantly associated to
AN [31]. The serotonin transporter gene locus (SLC6A4) has
a functional promoter polymorphism associated with
transcription efficiency [34] that has been associated with
anxiety-related traits [35]. Several studies with this
polymorphism do show statistically significant association
to AN [36, 371, while some studies fail to show association
[38, 391. A screen of the tryptophan hydroxylase locus
(TPH) did not identify any polymorphisms associated to AN
~401.
Six serotonergic candidate gene loci have been the
subject of publications in the AN candidate gene literature,
Le., the genes encoding the serotonin receptors lB, 2A, 2C
and 7, the serotonin transporter, and tryptophan hydroxylase,
Table I.
Catecholamine Candidate Gene Studies Through 2001
Polymorphisms at three dopaminergic genes have been
investigated for association to AN in the literature, Le.,
Serotonergic Candidate Gene Association Studies in AN Through 2001
Gene Locus
OMIM#
Chr
Poly.
Cn. N
Result
182131
6q13
Phe124Cys-
393
NS
182135
13q14-q21
-1438GiA
170
NS
+
1
1
78
~
I
Citation
Hinnev. '99
Ziegler, '99
81
226
S
Collier, '97
100
295
NS
Hinney, '97
152
150
NS
Campbell, '98
77
107
S
Sorbi, '98
68
69
S
Enoch, '98
45
45
NS
Karwautz, '01
109
107
S
Nacmias, '01
45
NS
Karwautz, '01
393
NS
Hinney, '99
312861
Xq24
HTR7
182137
1oq2 1-q24
Pro279Leu
SLC6A4
182138
17ql1.1-qI2
Promoter
90
NS
Sundaramurth, '00
VNTR
120
S
Di Bella, '00
67
148
NS
Fumeron, '01
67
358
S
Fumeron, '01
96
385
NS
Hinney, '97
128
142
NS
Han, '99
TPH
1
191060
111115.3-I1.4
C1095T
1
1
84
-
Price Foundation Anorexia Nervosa Dataset
Current Drug Targets CNS & Neurological Disorders 2003, Vol. 2, No. 1 43
DRD3, DRD4 and COMT (Table 11). Previously, these
genes have been associated to schizophrenia @RD3 receptor)
[41], novelty-seeking and substance abuse (D4 receptor,
inconsistent findings) [42], and OCD (COMT) [43]. The
published reports in the eating disorder literature do not
identify an association to AN at polymorphisms at these
genes in case:control studies [44, 45, 291. Two family-based
association studies using a functional substitution
polymorphism at COMT (Val1 58Met) present evidence for
association with AN, but in each study a different allele is
reported to be the risk allele, i.e., the high activity allele in
[46], and the low activity allele in the Klauck et al., personal
communication cited in [46]. A substitution polymorphism
at the P3-adrenergic receptor, previously associated with
obesity and related phenotypes [47], was not found to be
associated to AN [48].
traits [54], and a microsatellite marker linked to the UCP2/3
gene cluster on chrl lq13 has shown significant association
to AN [ S I , however the significance of this finding should
be evaluated by association analysis of variation at the genes
themselves. The estrogen receptor beta gene, a
polymorphism at which has been associated with ovulatory
dysfunction [56], has also been shown to be nominally
significantly associated to AN [57]. Finally, studies at the
major histocompatibility locus HLA-A have been
inconclusive [58, 591.
In summary, over twenty polymorphisms at sixteen
(N=l6) candidate genes have been evaluated for association
with the diagnoses of AN in the psychiatric genetic literature
(Tables I and 11). Association findings at the serotonin 2C
receptor and serotonin transporter loci has been shown to
reproducibly convey risk, however, the risk is modest, Le., a
RR of -1.4 fold and -1.25 fold, respectively. Why are the
results of most of these candidate gene association studies to
AN phenotype negative or equivocal despite the use of
neurobiological hypotheses to nominate candidates? The
fundamental reasons are 1) the very low prior probability of
any particular candidate gene being associated with any
complex disorder [60], due to the large number of genes
(>30,000) and sequence polymorphisms (>3,000,000)
known to exist in the genome [61], 2) the large number of
individuals required to detect the modest genotypic relative
risks expected in a complex disorder such as AN in
association and linkage designs [62], and the possibility that
the existing psychiatric diagnostic criteria of AN may not be
the best phenotype to identify genetic susceptibility factors
in the eating disorders [63]. In addition, samples are often
ascertained in widely variable and unsystematic ways, and
may differ with respect to a variety of factors influencing
outcome. These could include, for example, the presence of
comorbid traits and other modifying factors that influence
disease severity.
Neuroendocrine Candidate Gene Studies Through 2001
A series of appetite regulation and energy metabolism
candidate genes have been evaluated for association with AN
(Table 11). These candidate genes have been selected using
the abundant evidence that these genes are involved in the
control of appetite and feeding behavior [4]. A case:control
study has provided evidence that a two locus polymorphism
that includes a substitution polymorphism at the agouti
related protein locus is associated with AN [49]. DNA
polymorphisms at the following neuroendocrine loci were all
studied for associations with AN: leptin, the adipocyte
orexigenic hormone; melanocortin receptor-4, a receptor
regulating feeding with agonist NPY and antagonist AGRP;
the NPY Y5 receptor; and proopiomelanocortin, the locus
coding for corticotropins, lipotropins, melanotropins and
beta-endorphin. For all of the polymorphisms tested, no
statistically significant association was found with AN [50531. The uncoupling proteins UCP1, UCP2, UCP3 have
previously been associated with energy expenditure related
Table 11. Non-serotonergic Candidate Gene Association Studies in AN Through 2001
Locus
OMIM#
Chr
Poly.
Ca. N
Cn. N
DRD3
125451
3q13.3
Ser9Gly
39
42
NS
327
NS
Hinney, '99
NS
Hinney, '99
DRD4
126452
llp15.5
Exldel,Ex3VNTR
109
Exldel,Ex3VNTR
57 Trios
Ex3 VNTR
COMT
1
AGRP
1
6023 11
LEP
MCH4
116790
155541
18422
601693
llq13
ESRl
601663
14a
1
142800
1
6q21.3
Vall58Met
45
1
1
I
45
45
1
45
1
Citation
1
Bruins-Slot, '98
NS
Kanvautz. '01
NS
Kanvautz. '01
760G/A, 526GlA
145
244
S
Vi&, '01
830CiT
145
244
NS
Vink, '01
315
NS
Hinnev. '98
52
NS
Hinnev. '99
Campbell, '99
- 1387GIA
7a31.3
1
UCP
HLA-A
I
16q22
164160
1
22q11.2
Result
Vall03lle
115
1
51
1
D11 S911
170
150
S
DllS916
170
150
NS
Campbell, '99
1082GlA
50
96
S
Rosenkranz. '98
S
1 Biederman. '84
Bw16etal.
1
37
1
246
1
44
-
Current Drug Targets CNS & Neurological Disorders 2003, Vol. 2, No. I
AN-ARP DATASET COLLECTION AND ANALYSIS
In 1995, a multi-center collaborative study on the
genetics of AN and bulimia nervosa (BN) was organized by
Walter H. Kaye as Principal Investigator, with the financial
support of the Price Foundation of Geneva, Switzerland.
Data collection has been performed at multiple clinical sites,
with a group of clinically administered and self-report
questionnaires, using the Structured Interview for Anorexia
and Bulimia to make a DSM-IV diagnosis of AN [2]. A
sample of blood was collected from each individual for
genetic studies. The sample of eating disorder affected
relative pairs ascertained on a AN proband is known as the
Price Foundation Anorexia Nervosa Affected Relative Pair
dataset (AN-ARP) [2]. The affected relatives fulfill criteria
for DSM-IV anorexia nervosa (AN), bulimia nervosa (BN) or
eating disorder not otherwise specified (ED NOS). A second
sample of eating disorder affected relative pairs was
ascertained on a BN proband, collected, and is known as the
Price Foundation Bulimia Nervosa Affected Relative Pair
dataset (BN-ARP) [64]. Blood samples from parents have
been collected to provide additional genetic information for
gene mapping studies. The first ARP dataset collection was
completed in 1998 and the second was completed in 2000.
A Weber version 9 (Marshfield Medical Foundation Center
for Medical Genetics) whole genome linkage scan has been
applied to DNA samples from both datasets [2, 641. Several
studies of the behavioral, clinical and temperament
phenotypes in the AN-ARP dataset, as well as three linkage
analyses of the two genome scans, are extant [5,64-691.
Biognosis U.S., Inc.
Biognosis U.S., Inc. (“Biognosis”) was founded in 1999
to identify and characterize candidate genes in the Price
Foundation eating disorder datasets. The founding group of
scientific advisors included scientists from the Price
Foundation Collaborative Group and scientists with
expertise in biological psychiatry, psychiatric genetics,
candidate gene analysis and human molecular genetic
analysis. The scientific mission of the company was to
identify genes increasing susceptibility to eating disorders.
Biognosis created a molecular genetic analysis capability
based on Applied Biosystems (Foster City, CA) sequencing
and genotyping technology platforms and the
implementation of a Labvantage (Bridgewater, NJ)
Laboratory Information Management System (LIMS).
Biognosis outfitted a laboratory for sequencing and
genotyping and implemented a LIMS in early 2000.
Biognosis U.S., Inc. was wound up in December of 2001.
The molecular genetic analysis capability developed in that
period was used to investigate candidate genes in the Price
Foundation eating disorder datasets and is described below.
Development of a Molecular Genetic Analysis Capability
Biognosis implemented the following molecular genetic
methods in its Gaithersburg, MD laboratory facility to
investigate the Price Foundation AN-ARP and BN-ARP
datasets. Blood and DNA specimens were received, and
Bergen et a,!
tracking of these samples was initiated within the LIMS.
DNA from blood samples from individuals whose DNA was
not already available was extracted using a standard high-salt
extraction procedure. Newly extracted DNA and other DNA
samples received into the laboratory were quantified by
measurement of UV absorbance at 260 nm. DNA was
diluted, aliquoted, and normalized to 10 ng/uL based on this
quantification information and assessment of a test PCR.
Aliquots were first plated onto master 96-well plates and
subsequently onto working plates (50ng/well), then
lyophilized for later use in genotyping assays. Blood
samples received as duplicates were stored appropriately and
extracted at a later date when it was determined that DNA
was needed.
The selection of candidate genes to investigate in the
Price Foundation AN-ARP dataset was ’based on
neurobiological and positional hypotheses [2-51.
Characterization of these candidate genes and optimization of
genotyping assays for selected genetic variation within these
genes was based on bioinformatic characterization of
sequence and polymorphism information from public
databases. Genomic and mRNA sequence and polymorphism
information was downloaded from public databases and
obtained from the biomedical literature and assembled to
form a genomic sequence contig with annotation at structural
and variable positions. Information concerning variation at
these candidate loci was incorporated into the LIMS and
variation was prioritized for validation and genotype assay
development. In cases where genomic variation at a
candidate gene was not available in public databases or the
literature, or the information concerning the variation was
incomplete, a portion of the gene or the entire gene, was
sequenced in order to identify or validate variable sites using
PCR-based DNA resequencing. PCR primers were designed
to produce 300-600 bp products that were resequenced in 32
unrelated CEPH individuals using Applied Biosystems
(Foster City, CA) sequencing reagents, automated
sequencers, and sequencing analysis software. This
additional information was incorporated into the LIMS and
used to select variable sites (mostly single nucleotide
polymorphisms or SNPs) to genotype in the Price
Foundation AN-ARP and control datasets. Decisions on
which candidate gene SNPs to genotype were based on the
criteria of a priori findings in the literature, allele frequency,
possible substitution effects, and location of the
polymorphism within a candidate gene, derived from the
bioinformatic analysis of public and Biognosis generated
sequence information.
Biognosis utilized four different genotyping platforms:
TaqManTM S’exonuclease, fluorescent primer extension
genotyping (SNap-ShotTM), short tandem repeat genotyping
by fluorescent PCR size determination using gel
electrophoresis, and PCR-RFLP analysis. The majority of
genotyping assays were designed as TaqMan S’exonuclease
assays. For the S’exonuclease assays, dual-labeled probes
and primers were chosen using ProbeITY (Celadon
Laboratories, College Park, MD) and synthesized by
Applied Biosystems. Assays were optimized using standard
conditions [70], by using control DNAs with known
genotypes for a single nucleotide polymorphism (SNP), and
Price Foundation Anorexia Nervosa Dataset
by modifying assay conditions until an assay produced those
known genotypes accurately, as measured by the criteria of
completion and concordance in a duplicate sample of CEPH
DNAs. Once an assay was optimized, it was applied to case,
family and control DNA samples. Genotype determination
was conducted manually using Applied Biosystems s o h a r e
on the respective Applied Biosystems instrument (Sequence
Detector 7700 or Automated Sequencer 377XL). For most
assays, a verification plate consisting of a 17% duplication
of the appropriate data set and control group samples was
genotyped at the same time in order to assess genotype
concordance rate. Genotyping error rate was assessed by the
use of no template (no genomic DNA) and known genotype
controls, and the criteria of completion rate, concordance
rate, and Hardy Weinberg Equilibrium (HWE) contingency
analysis.
Development of a Informatics Infrastructure
From October through December of 1999, Biognosis
investigated existing LIMS vendors and software
applications for integration into the laboratory. Biognosis
executed a license with a Labvantage Solutions for its
Sapphire LIMS software in December of 1999 and
implemented a LIMS throughout 2000. The Biognosis
LIMS was based on an Oracle database, extensive use of
VBScripts and built-in client side workflows. This LIMS
was designed and constructed to assist the laboratory in three
major areas: sample and reagent tracking, clinical,
bioinformatic and genotype databasing, and data analysis
and reporting.
In the sample tracking portion of the Biognosis LIMS,
users tracked samples and information about those samples
throughout all stages of laboratory use. This included
sample tracking from multiple sites into multiple storage
sites, and aided in the selection of samples to be cued for the
sample extraction workflow, and queries based on the current
stage of the samples. The LIMS also tracked individual
samples as they were transferred to aliquots on laboratory
plates. Plate creation and tracking in any laboratory
workflow followed the laboratory protocols that created and
used various types of plates, including master plates,
working plates, and working dilution plates. Groups of
plates, for example, could be defined as an assay and
selected for further analysis in a particular workflow. These
workflows included extraction, plate creation, plate
replication, PCR and genotyping assay set-up workflows.
In the databasing portion of the Biognosis LIMS,
clinical, bioinformatic and genotypic data attributes were
tracked and recorded. The LIMS managed clinical data, i.e.,
phenotype and pedigree data of the AN and BN ARP
datasets. The LIMS performed candidate gene databasing
including the locus and polymorphism data storage, primer
and probe tracking and data storage, as well as information
and analysis related to genetic markers. This enabled
workflows to be incorporated allowing for automated data
entry for TaqManTMexports. For TaqManTM,users initiated
a menu driven process to select the desired file, then the
workflow imported the data, associated it with individual
samples, and attached the raw data file to the plate.
-
Current Drug Targets CNS & Neurological Disorders 2003, VoL 2, No. 1 45
The Biognosis LIMS also assisted in data analysis of
genotypes and association between genotypes and
phenotypes. The LIMS had routines that automatically
calculated dropout rates as a count of failed genotypes,
flagged plates based on % completion per plate, measured
discordance between duplicated samples, tested HWE and
performed contingency table (association) analysis on alleles
and genotypes, after the user selected a series of plates to be
analyzed. The LIMS also enabled reporting (exporting) of
data in various formats for a number of external analytical
programs, e.g., Linkage, TDT, EM, Arlequin and Excel
formats as well as producing statistical reports. These
statistical reports ranged from simple reports, such as
reporting the numbers of plates in the LIMS used for each of
the various genotyping assays, to contingency analyses of
genotypes and phenotypes. Candidate gene polymorphisms
that were identified as nominally associated by contingency
analysis in this manner were further investigated using
statistical analysis as described below using data exported
from the LIMS.
Statistical Analysis of Candidate Gene Genotypes
Multi-locus genotypes were exported from the LIMS and
assembled in a Visual Basic utility and resulting multilocus
genotype counts were used to estimate intragenic and
intergenic painvise linkage disequilibrium using likelihood
ratio tests with empirical significance testing using Arlequin
[71]. The significance of differences in genotype and allele
frequencies at the polymorphic loci between the AN and
control samples was evaluated using contingency analysis in
SAS. In case of expected cell frequency 15,the p-value was
based on Fisher's exact test. The empirical significance of
haplotype frequency differences between AN and control
samples was evaluated using the nonparametric heterogeneity
statistic (T5) in the program EH+ using 10,000
permutations [72]. In order to maximize the power to detect
transmission disequilibrium at individual SNPs, FBAT was
used for transmission disequilibrium analysis (TDT) [73].
Power and the required sample size were evaluated using chisquare distribution properties and, for the TDT, the pedigree
structures. A p value of 50.05 is described in all analyses as
indicating a statistically significant result, while p values
20.05 and <0.10 are described in all analyses to indicate a
trend towards statistical significance.
Control DNA Samples
DNA samples collected from a group of EuropeanAmerican females (EAF) were used as control DNA samples
for genotype and allele frequency comparisons in the
case:control design. The EAF sample consists of EuropeanAmerican females (N=125), recruited through
advertisements, screened to exclude obese individuals
(>120% ideal weight) and to exclude lifetime criteria for
Axis I disorders as assessed by clinical administration of the
Structured Clinical Interview for DSM-111-R (SCID) [74].
Two publicly available sets of DNA samples were utilized
for SNP discovery and validation and for quality control
samples of known genotype. These two sets of samples were
the Centre EtudC Polymorphisme Humaine (CEPH) DNA
46
-
Current Drug Targets CNS & Neurological Disorders 2003, VoL 2, No. I
samples and the Coriell Caucasian Human Diversity Panel
(COR), obtained from the Coriell Cell Repositories. CEPH
DNA was used primarily for resequencing and as control
DNAs for genotype assay development and for genotyping.
The CEPH DNA samples are DNA samples extracted from
cell lines of a set of American, French and Venezuelan
pedigrees collected for the purpose of recombination
mapping by a collaboration organized by Jean Dausset of
CEPH. The CEPH samples used at Biognosis consisted of
unrelated individuals. COR DNA samples were used as an
additional DNA sample for various quality control purposes.
The COR sample consists of 100 DNA samples (N=49
females, N=51 males) from cell lines maintained at the
Coriell Cell Repository from self-identified Caucasians.
Candidate Gene Analysis in the AN-ARP Dataset
Biognosis' molecular genetic analysis strategy was to
investigate candidate genes whose products are involved in
monoamine and neuropeptide neurotransmission and in the
central nervous system control of appetite regulation. These
genes are nominated as candidates in AN through studies
investigating CSF levels of monoamine and neuropeptides,
in models of AN which emphasize behavioral reward
reinforcement and in animal models of anxiety, appetite
regulation and eating behavior. The following general
hypotheses regarding the biological basis of AN were
applied to nominate candidate genes for investigation. A
leading hypothesis is that serotonergic dysfunction, revealed
in studies of CSF serotonergic metabolite differences
between eating disorder patients before and after recovery,
and also between recovered eating disorder patients and
controls, increases vulnerability to eating disorders [75].
Another leading hypothesis is that the stress of selfstarvation and exercise, by increasing the release of
corticotropin releasing factor from the hypothalamus,
activates dopaminergic, adrenergic and opioidergic pathways
[36]. Indeed, two studies of concentrations of opioid
Bergen et aL
peptides have shown differences in individuals with AN
versus controls [77, 781. Those individuals vulnerable to
reward reinforcement might, according to this hypothesis,
become addicted to the reward produced by the activation of
the Hypothalamic Pituitary Adrenal axis [79]. Another class
of hypotheses concerns central nervous system (CNS)
control of food intake, which resides primarily within the
hypothalamus and has been categorized into gene products
that increase food intake (orexigenic) and those that decrease
food intake (anorexigenic) [4]. Many neuropeptides and
receptors are involved in this feedback loop, and several
reward reinforcement candidates are also involved in the
appetite regulation. Finally, a positional hypothesis derived
from a linkage scan of the AN-ARP dataset helped to
prioritize several candidates for molecular genetic
investigation. Specifically, genome-wide linkage analysis of
the entire AN-ARP dataset provided only modest evidence
for linkage when all DSM-IV eating disorder diagnoses were
included in the affected sample [5]. However, linkage
analysis in a subset of 37 families with at least one affected
relative pair concordantly affected with a DSM-IV diagnoses
of restricting AN (a subtype of AN), provided suggestive
evidence for linkage with a maximum multipoint NPL score
of 3.45 found on chromosome 1 ~ 3 3 - 3 6at marker DlS3721,
located at -72.6cM on the Marshfield linkage map [5].
Analysis of OPRDl and HTRlD
Results from two examples of candidate genes with
statistically significant association to AN phenotype in the
Price Foundation AN-ARP dataset, i.e., the delta opioid and
serotonin 1D receptor, OPRDl and H T R l D [SO], are
reviewed below. OPRDl and HTRlD are candidate genes for
AN nominated by multiple hypotheses: positional (both
genes are located on chromosome 1p33-36, a region linked
to AN [5]; the anxiety and appetite modulation functions of
the OPRDl gene product; and the autoreceptor function of
the HTRlD gene product. These two candidate loci were
Table 111. HTRlD and OPRDl SNPs Genotyped in the AN-ARP Dataset
SNP'
Allele 1
Allele 2
SNP IDZ
Coding region
Source
HTRID(-l123T>C)
T
C
SNP000083015
No, 5' of coding
Reference 80
31.3
HTRlD(-628T>C)
T
C
SNP000083091
No, 5' of coding
Reference 80
14.1
HTRl D( 1080C>T)
C
T
SNP000006432
Yes, silent
Reference 83
9.4
HTRlD(Zl90A>G)
A
G
SNP000080270
No, 3' of coding
Reference 80
65.6
OPRDl(BOT>G)
T
G
SNP000063484
Yes, F27C
Reference 82
OPRDl(8214T>C)
T
C
SNPOOlO26473
No, IVS 1
TSC0110129
OPRDl(23340G>A)
G
A
SNP000085600
No, IVS 1
TSC0110127
OPRDl(4782 1A>G)
A
G
SNP000066643
No, IVS 2
Reference 80
~~~
OPRDl(5 1502A>T)
A
T
From the ATG of the candidate gene genomic sequence.
'HGVBASE at http://hgvbase.cgb.ki.se/.
3Percent allele 1 from 32 unrelated CEPH DNAs obtained by resequencing.
SNPOOOO66640
No, 3' of coding
TSCOl10133
~ 1 3
41.1
Price Foundation Anorexia Nervosa Dataset
identified as positional candidates through a search of
unfinished BAC sequences from the high throughput
genome sequencing (HTG) section of Genbank [81] using
the sequence of chrlp33-36 microsatellite markers. OPRDl
and H T R l D reside a t 55.7cM133.5Mbp and
49.0cM/25.7Mbp, -23cM and 17cM distal to the linkage
peak at 72.6cM [SO], respectively. However, OPRDl and
HTRlD are not in significant linkage disequilibrium with
each other in the AN sample [80]. Therefore, for the
purposes of association analysis, these two genes can be
treated as two distinct genetic targets.
Sequence Variation at OPRDl and H T R l D SNPs
The OPRDl locus contains three exons of 51,551 base
pairs with 1,173 base pairs of cDNA sequence and the
HTRlD locus contains a single exon with 1,134 base pairs
of cDNA sequence. In order to validate previously reported
SNPs and to identify novel SNPs, portions of both loci
were resequenced in 32 CEPH individuals: 2.8Kb of the
HTRlD locus (from -1165bp 5’ to +1497bp) and 4.18kb
portion of the genomic sequence surrounding exon 2 of
OPRDl (from IVS1-1860 to IVS2+1996) [SO]. The
resequencing of 32 CEPH samples at OPRDl and HTRlD
identified one de novo OPRDl SNP and four de novo
H T R l D SNPs and validated one previously reported
HTRlD SNP [82]. Nine TaqManTMgenotyping assays were
developed, four at HTRlD and five at OPRD1, from d e
novo, previously reported and TSC in silico SNPs (Table
111) [SO]. Average genotype completion and concordance
rates were 95% and 98.2% for the AN proband sample and
88% and 98.6% for the EAF control sample, respectively
[go].
-
Current Drug Targets CNS & Neurological Disorders 2003, VoL 2, No. 1 47
suggest that the OPRDl locus may be involved in
susceptibility to AN, however, this association should be
investigated in additional samples using both case:control
and family-based designs in order to confirm the results
from the Price Foundation AN-ARP dataset.
Association of H T R l D SNPs to AN
One H T R l D SNP, HTRlD(lOSOC>T), of four
genotyped in the Price Foundation AN-ARP dataset (Table
IV), was observed to be significantly associated to AN
(p=O.Ol, both genotypic and allelic) with a OR of 2.63
(95CI 1.21-5.75) [SO]. Significant HTRlD SNP haplotype
frequency heterogeneity between the AN proband and EAF
samples was observed with the three pair-wise haplotypes
containing the HTRlD( 1080C>T) SNP (p<0.04), but not
with the haplotype containing all four HTRlD SNP alleles
[SO]. While significant transmission disequilibrium was
observed at three HTRlD SNPs, it was not observed at
HTRlD(1080C>T) [SO], probably because of the lack of
power of the TDT with low minor allele frequencies [84].
These three H T R l D SNPs (-1 123T>C, -628T>C and
2190A>G) were observed to be in complete linkage
disequilibrium with each other, while the HTRlD 1080C>T
was not observed to be in complete linkage disequilibrium
with the other three [SO]. As the HTRlD(lOSOC>T) SNP is
a silent mutation and the three remaining SNPs are 5’ or 3’
of the coding region, it is possible that these SNPs may be
associated with differences in HTRlD gene expression or in
linkage disequilibrium with other polymorphisms at the
H T R l D locus. Again, this association should be
investigated in additional samples using both case:control
and family-based designs in order to confirm the results
from the Price Foundation AN-ARP dataset.
Association of OPRDl SNPs to AN
Linkage of OPRDl and HTRlD to AN
-Three of five OPRDl SNPs genotyped in the Price
(Table
IV),
Foundation
AN-ARP
dataset
OPRD1(8214T>C),
OPRD1(23340A>G),
and
OPRD l(4782 lA>G), were observed to be significantly
associated to AN (p=<0.05) [SO]. The strongest evidence
was found for OPRD1(47821A>G) (p=O.Ol) with a OR of
1.64 (95% CI 1.1 1-2.44), allele 2 to allele 1 [80].
Significant differences in OPRDl SNP haplotype
frequencies between the AN proband and EAF samples was
observed
with
OPRDl
SNP
haplotype
(8214T>C)1(47821A>G) (p=0.04) [80]. OPRDl 80G allele
frequencies determined in the EAF sample (minor allele =
0.1 1) were not different to the previously reported frequency
(minor allele = 0.12) in European-Americans [83] and there
is no significant allele frequency difference between the AN
proband and EAF control samples at this polymorphism
(p=0.94). Significant allele frequency differences among six
continental population groups have been previously observed
at this polymorphism [83]. This suggests that the AN
proband and EAF samples studied here may be ethnically
matched at a continental population level. This study also
found a trend towards transmission disequilibrium at two
OPRDl SNPs (4782 1A>G and 5 1502A>T)(p=0.06); these
two O P R D l S N P s exhibited nominal linkage
disequilibrium with each other (p=0.034) [SO]. These results
After incorporating the OPRDl and HTRlD SNPs in the
linkage analysis using the same sample of N=37 families
with concordantly restricting AN affected sib pairs [5], the
strength and significance of the linkage signal previously
observed was increased substantially, by 0.46 Z and an order
of magnitude in p value to 3.91 and p=0.00005, respectively
[SO], from the previous analysis [5]. However, OPRDl and
H T R l D are >16cM from DlS3721 and the correlation
between allele sharing at the candidate SNPs and chrlp
microsatellites, while substantial, does not account for the
complete linkage signal [SO]. Note that the addition of the
candidate gene SNPs did not reduce the width of the linkage
peak and introduced a secondary linkage peak with a NPL
score of 3.05 at OPRDl [80].
Power to Detect Observed Allelic Effects
The estimated total number of cases and controls
necessary to have a 50% chance to detect a given allelic
effect (odds ratio) at a given minor allele frequency using the
case:control (50:50) design with a categorical phenotype and
allele counts at a biallelic polymorphism was calculated. To
detect an odds ratio of 1.6 with a minor allele frequency of
48
Current Drug Targets - CNS & Neurological Disorders 2003, Vol. 2, No. I
SNP
N
x2
p
OR
Bergen et aL
non-significant findings, despite the careful selection of
neurobiological candidate genes. This also suggests that a
significantly larger sample will be required to attempt to
replicate the candidate gene associations described here.
Consideration of the severity of the AN diagnosis will also
be important in defining a sample for follow-up of these
findings, as the AN-ARP probands are chronically affected
individuals from multiplex families and are likely to be
more severely affected than singleton affecteds.
(95%CI)
HTRlD(-1123T>C)
Follow-up to Price Foundation AN-ARP Findings
I
Alleles
546
2.64
.10
Genotypes
262
3.65
.17*
Alleles
524
.01
.94
Genotypes
270
3.84
0.15
Alleles
540
4.00
0.046
Genotypes
258
7.05
0.03
Alleles
516
6.32
0.01
Genotypes
268
4.14
0.13
Alleles
536
3.51
0.06
1.37
.94-1.99
.98
.55-1.76
.68
.47-.99
0.61
.41-.90
OPRD 1(80T>G)
PRD l(82 14TzC)
Genotypes
Alleles
PRD l(23 340A>G)
OPRD1(47821A>G)
I
LPRDl(5 1502A>T)
.70
0.48-1.02
42%, that of the OPRD1(47821A>G) SNP [SO], it is
necessary to have -300 individuals in a case-control study.
Substantially more individuals will be required to have a
50% chance of detecting the HTRlDl080C>T SNP effect,
given an observed minor allele frequency between 10 and
15%, -1500 individuals. Thus, it is apparent why the
modest number of cases and controls in the published eating
disorder candidate gene literature has generally resulted in
Several factors related to candidate gene association study
design will influence association study results, whether the
candidate gene association study is directed towards
discovery or validation of association between candidate
gene polymorphisms and AN. As candidate gene association
analysis will not be limited to a single locus or
polymorphism, but will be an ongoing process in which
novel candidate regions, genes and polymorphisms are tested
as they are nominated, statistical correction with a more
significant a level will be required, just as in linkage
analysis [85]. Exhaustive and systematic association studies
of chromosomal regions nominated by linkage analysis may
require the genotyping of hundreds of single nucleotide
polymorphisms (SNPs), even in small genomic regions, in
order to move from linkage analysis to the identification of a
susceptibility gene, e.g., [86], increasing sample size
requirements. In the case of whole-genome association scans,
an a value of 5 X
has been suggested by several
authors [62, 871, but it is unlikely that 500,000 - 1,000,000
markers will ever be used in practice. Genotyping of several
SNPs per gene will allow haplotypes to be estimated
computationally, and thus most candidate gene haplotypes
will be highly informative. An approach using SNPs
primarily located at or within genes will increase the
probability of identifying markers in linkage disequilibrium
with functional polymorphisms [62]. Both of these factors
will tend to reduce required sample sizes. With a relative
risk estimate of 11 for AN [15], one of a number of
oligogenes may have a genotypic risk ratio (GRR) of 1.5 to
2.0. If preliminary statistical analysis suggests that more
than one allele is associated with increased risk, e.g., as for
hemochromatosis [88], these alleles could be lumped
together to create an at-risk group of alleles. However,
without a priori knowledge of allelic effects, multiple alleles
at a locus may increase the required sample size (depending
upon frequencies and GRRs of the alleles). The above
considerations suggest that in order to pursue large-scale
association studies with AN phenotypes using either the
case:control or the trio design, a AN sample size several
times greater than the Price Foundation AN-ARP dataset is
required.
One approach towards estimating required sample sizes
suggests that a sample of 500-800 trios with a proband
affected by AN and one or two parents would provide a
sample powerful enough to pursue the range of association
strategies outlined above [62, 841. This sample size would
be sufficient to detect GRRs -2.0 in both discovery and
validation oriented candidate gene association studies,
including the systematic analysis of genomic regions
-
Price Foundation Anorexia Nervosa Dataset
nominated b y linkage analysis [ 5 , 64, 691. However, if
genetic susceptibility to eating disorders reflects the
operation of hundred of genes of small effect, even largescale studies will be underpowered. Fortunately, the Price
Foundation’s scientific advisors anticipated the necessity for
the collection of a large association dataset in 1999 and
made a recommendation for such a collection. This
recommendation resulted in the collection of a third Price
Foundation Collaborative Group clinical sample known as
the Price Foundation AN Trio/Control Women dataset in
2000-2002. This sample is composed of a sufficient number
of AN cases, parents of cases, and unrelated control women,
to enable both the case:control and trio designs to be used in
candidate gene association studies motivated by linkage and
association findings identified in the Price Foundation ANARP dataset.
ACKNOWLEDGEMENTS
The authors gratefully acknowledge the financial support
of the Price Foundation.
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