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 firstname.lastname@example.org 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.  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.  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.  lacked a healthy, normal weight, unrelated control population. To try and replicate the data of Hinney et al.  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). 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