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American Journal of Medical Genetics (Neuropsychiatric Genetics) 96:53–55 (2000)
Analysis of the Serotonin Transporter Gene Linked
Polymorphism (5-HTTLPR) in Anorexia Nervosa
D. Sundaramurthy,1 L.F. Pieri,1,2 H. Gape,1 A.F. Markham,1 and D.A. Campbell3*
1
Molecular Medicine Unit, University of Leeds, St. James’s University Hospital, Leeds, United Kingdom
Yorkshire Centre for Eating Disorders, Seacroft Hospital, York Road, Leeds, United Kingdom
3
SmithKline Beecham Pharmaceuticals, Essex, United Kingdom
2
INTRODUCTION
Previous studies have demonstrated aberrant expression of serotonin in individuals
with an eating disorder. Given this the serotonin transporter gene (5-HTT) is a strong
candidate to contribute to the genetic component of the aetiology of eating disorders.
To determine the role of this particular gene
in the susceptibility to anorexia nervosa
(AN) we have examined a tandemly repeated sequence close to the promotor region of the 5-HTT gene, which is represented by a long (L) and short (S) variant.
Previous studies have shown that the transcriptional activity of the 5-HTT gene differs
significantly between these two alleles. A
group of 138 Diagnostic and Statistical
Manual of Mental Disorders (DSM-IV) criteria AN patients and 90 controls were genotyped at the 5-HTT gene linked polymorphism (5-HTTLPR). Statistical analysis
showed no significant difference in allele or
genotype frequencies between the two
groups. These data suggest that there is no
association between 5-HTTLPR genotype
and susceptibility to AN, in our population.
Am. J. Med. Genet. (Neuropsychiatr. Genet.)
96:53–55, 2000. © 2000 Wiley-Liss, Inc.
KEY WORDS: anorexia nervosa; serotonin;
serotonin transporter; polymorphism; association
analysis
Contract grant sponsor: Northern and Yorkshire Regional
Health Authority; Contract grant sponsor: Leeds Community and
Mental Health NHS Trust; Contract grant sponsor: Royal Society; Contract grant sponsor: Wellcome Trust.
*Correspondence to: D. Campbell, Genetic Technologies,
SmithKline Beecham Pharmaceuticals, New Frontiers Science
Park (North), Third Avenue, Harlow, Essex, UK.
E-mail: david a campbell@sbphrd.com
Received 17 December 1998; Accepted 29 July 1999
© 2000 Wiley-Liss, Inc.
The human serotonin transporter gene (5-HTT) localises to chromosome 17q11-12 and encodes a transmembrane protein that functions in the reuptake of
serotonin [5-HT; Ramamoorthy et al., 1993]. By facilitating 5-HT reuptake, 5-HTT protein is thought to be
involved in the regulation of some anxiety-related
traits [Risch et al., 1992]. Deregulation of 5-HTT protein function has also been implicated in the pathophysiology of a number of neuropsychiatric disorders
including depression [Owens et al., 1994], schizophrenia [Joyce et al., 1993], neurodegeneration [Meltzer et
al., 1981], and eating disorders [Di Bella et al., 1998].
Recently Heils et al. [1996] reported an allelic variation in the 5-HTT gene in a tandemly repeated sequence ∼1 kb upstream of the transcription initiation
site. This polymorphism consists of a long variant (L)
composed of 16 repeat elements or a short variant (S)
generated by a deletion of 44 bp. These two alleles are
found to modulate 5-HTT gene expression, with the
S-allele being associated with reduced transcriptional
efficiency of the 5-HTT gene promoter, resulting in decreased protein production and, by inference, decreased 5-HT reuptake [Heils et al., 1995, 1996].
Abnormal levels of serum serotonin have previously
been described in anorexia nervosa (AN) [Kaye et al.,
1991]. A recent study by Hinney et al. [1997] reported
no association between allele or genotype frequencies
at the 5-HTTLPR polymorphism and weight regulation. Further comparisons of allele and genotype frequencies in their AN population failed to demonstrate
an association between this region of the genome and
genetic susceptibility to AN. However, the numbers of
patients in each group were relatively small (n ⳱ 55 for
AN) and the study by Hinney et al. [1997] lacked a
healthy, normal weight, unrelated control population.
To try and replicate the data of Hinney et al. [1997] and
clarify the relative importance of the serotonin transporter gene polymorphism as a possible etiological factor in AN, we performed a case-control, association
study in a group of 90 controls and 138 anorexics. Ethical approval for this study was obtained from Leeds
(East) Medical Research (Ethics) committee. Written
informed consent was obtained from all individuals.
54
Sundaramurthy et al.
TABLE I. 5-HTTLRP Genotype and Combined Genotype Distribution (Frequency) in
Anorexia Nervosa Patients and Controls
Genotype
Group
AN patients
Controls
Combined genotype
Long/Long
Long/Short
Short/Short
Long/Long
-/Short
40 (0.29)
34 (0.38)
63 (0.46)
40 (0.44)
35 (0.25)
16 (0.18)
40 (0.29)
34 (0.38)
98 (0.71)
56 (0.62)
MATERIALS AND METHODS
Subjects
A cohort of 138 sufferers (DSM-IV criteria) was obtained from the clinics of Dr. Pieri. Sufferers were all
British Caucasian females and diagnosis was made on
the basis of a structured interview. Mean age of disease
onset of this group was 18.1 years with a minimum average body mass index (BMI) of 13.73 kg/m2 during
illness. The control cohort of 90 British Caucasian females was obtained from a number of sources. Mean
age of the controls was 30.28 years with an average BMI
of 22.02 kg/m2. The latter were screened for a personal
or immediate family history of an eating disorder or
other psychiatric illness.
Polymerase Chain Reaction (PCR)
PCR across the 5-HTT gene linked polymorphic region (5-HTTLPR) was performed using oligonucleotide
primers corresponding to nucleotide position −1416 to
−1397 (5⬘-dGGCGTTGCCGCTCTGAATGC) and −910
to −888 (5⬘-dGAGGGACTGAGCTGGACAACCAC) generating 484 (S) or 528 (L) bp. fragments [Lesch et al.,
1996]. Successful amplification was achieved using an
initial denaturation step at 95°C for 5 min followed by
35 cycles of 95, 61, and 72°C for 30 sec each and a final
extension step at 72°C for 10 min. The 20 ␮L reaction
consisted of 10 mM Tris-HCl (pH 9.0), 50 mM KCl, 1.5
mM MgCl2, 0.5 ␮M each primer, 200 ␮M dNTPs, and
0.25 units Taq DNA polymerase (Promega). The amplified fragments were separated on a 2% agarose gel
(Flowgen) and bands visualised by ethidium bromide
staining and ultraviolet transillumination.
Statistical Analysis
Statistical analysis was performed using the Clump
program to identify any differences between allele or
genotype frequencies in our patients and controls
[Sham and Curtis, 1995]. This program constructs a
standard 2-by-n contingency table where n is the number of alleles at the test marker. The program then
generates a ␹2 value for this table called T1 (reported
here) and three further values T2, T3, and T4. Creating
a table in which all columns containing small numbers
are clumped together generates T2. T3 is generated by
comparing each column in turn with all other columns
clumped together to generate N-1 2-by-2 contingency
tables and tests the hypothesis that there is one particular column that has values deviating from the expected. The highest ␹2 value is then reported as T3.
Finally, clumping the 2-by-n table to produce a single
2-by-2 table that gives the largest ␹2 value generates T4.
For each of these values the program also performs a
Monte Carlo simulation within the constraints of the
original data set and returns a value corresponding to
the number of times the simulation produces a ␹2 value
equal to or greater than the actual value from the study
data set. This value allows a good estimation of the
true significance of the results.
RESULTS AND DISCUSSION
The genotype and combined allele frequencies of the
5-HTTLRP in our patients and controls is shown in
Table 1. Both patients and controls showed HardyWeinberg equilibrium for genotype distribution (data
not shown). Although the frequency of the short allele
(S) was higher among patients than controls (0.48 vs.
0.40), this was not statistically significant (p ⳱
0.1287). Allele and genotype frequencies in the AN patients vary slightly from those previously published
[Hinney et al., 1997]; however, these are not statistically significant (L-allele 0.48 vs. 0.44; S-allele 0.52 vs.
0.56; L/L-genotype 0.29 vs. 0.30; L/S-genotype 0.46 vs.
0.53; S/S-genotype 0.25 vs. 0.16).
It has been shown previously that the short allele (S)
reduces the transcriptional efficiency of the 5-HTT
gene to an extent where the 5-HT uptake is halved
compared with that of the L variant [Lesch et al.,
1996], and that there is no phenotype difference between heterozygous and homozygous forms of the short
allele. Hence we considered the S-allele as “dominant”
and compared the combined frequency of L/S and S/S
genotypes with the L/L genotype. Although the frequency of the combined genotypes was slightly higher
among patients than controls, again no overall significant difference in genotype frequency was observed (p
⳱ 0.1102).
Although the human 5-HTT gene represents a good
candidate for involvement in the genetic susceptibility
to AN, our preliminary results suggest that 5-HTTLRP
does not play a role in the genetic predisposition to AN
in this population. However, further studies, including
family-based control studies, are still warranted to rule
out the effects of population stratification. It is possible
that another polymorphism, not in linkage disequilibrium with 5-HTTLRP, may still form part of the genetic predisposition to AN.
REFERENCES
Di Bella D, Cavallini MC, Riboldi C, et al. 1998. Genetic studies in eating
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Neural Transm (Gen Sect) 102:247–254.
5-HTTLPR and Anorexia Nervosa
Hinney A, Barth N, Ziegler A, et al. 1997. Serotonin transporter genelinked polymorphic region: allele distribution in relationship to body
weight and in anorexia nervosa. Life Sci 61:PL295–303.
Joyce JN, Shane A, Lexow N, et al. 1993. Serotonin uptake sites and serotonin receptors are altered in the limbic system of scizophrenics.
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Lesch KP, Bengel D, Heils A, et al. 1996. Association of anxiety related
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55
Meltzer H, Arora R, Baber R, et al. 1981. Serotonin uptake in blood platelets of psychiatric patients. Arch Gen Psych 38:1322–1329.
Owens MJ, Nemeroff CB. 1994. Role of serotonin in the pathophysiology of
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