Original Research Article

Molecular Psychiatry (2003) 8, 933–936. doi:10.1038/sj.mp.4001365

Serotonin transporter missense mutation associated with a complex neuropsychiatric phenotype

N Ozaki1, D Goldman2, W H Kaye3, K Plotnicov3, B D Greenberg4,5, J Lappalainen6, G Rudnick7 and D L Murphy8

  1. 1Department of Psychiatry, Fujita Health University School of Medicine, Toyoake, Aichi, Japan
  2. 2Laboratory of Neurogenetics, NIAAA, NIH, Rockville, MD, USA
  3. 3Department of Psychiatry, University of Pittsburgh Medical Center, Western Psychiatric Institute & Clinic, Iroquois Building, Pittsburgh, PA, USA
  4. 4Butler Hospital, Providence, RI, USA
  5. 5Department of Psychiatry, Brown University School of Medicine, USA
  6. 6Yale University School of Medicine, VA Connecticut Healthcare System, Psychiatry 116A2, West Haven, CT, USA
  7. 7Department of Pharmacology, Yale University School of Medicine, New Haven, CT, USA
  8. 8Laboratory of Clinical Science, MSC 1264, NIMH, NIH, Bethesda, MD, USA

Correspondence: Dr DL Murphy, Laboratory of Clinical Science, Building 10, Room 3D41, MSC 1264, 10 Center Drive, NIMH, NIH, Bethesda, MD 20892-1264, USA. E-mail: dm30h@nih.gov

Received 29 January 2003; Revised 7 April 2003; Accepted 16 April 2003.



Two common serotonin transporter (SERT) untranslated region gene variants have been intensively studied, but remain inconclusively linked to depression and other neuropsychiatric disorders. We now report an uncommon coding region SERT mutation, Ile425Val, in two unrelated families with OCD and other serotonin-related disorders. Six of the seven family members with this mutation had OCD (n=5) or obsessive-compulsive personality disorder (n=1) and some also met diagnostic criteria for multiple other disorders (Asperger's syndrome, social phobia, anorexia nervosa, tic disorder and alcohol and other substance abuse/dependence). The four most clinically affected individuals—the two probands and their two slbs—had the I425V SERT gene gain-of-function mutation and were also homozygous for 5'-UTR SERT gene variant with greater transcriptional efficacy.


genetics, obsessive-compulsive disorder, autism, anorexia, constitutive activation

Obsessive-compulsive disorder (OCD) occurs in 1–3% of the US population and is among the ten leading causes of disability worldwide.1 Its etiology is unknown, although family members are at a seven-fold higher risk than control.2 As OCD differs from other neuropsychiatric disorders that are treated effectively with multiple drugs in responding only to serotonin transporter (SERT) antagonists, we and others have been examining SERT gene variants in this disorder.3

The human SERT gene, SCL6A4, has been cloned and sequenced; it maps to chromosome 17 q11.1–q12.4,5,6 Like other Na+- and Cl--coupled monoamine transporters, it is organized in 14 exons that yield a protein with 12 membrane spanning domains.7 Mice with a deletion of one or two SERT gene alleles exhibit profound alterations in neurochemistry, behavior, body weight, drug responses and thalamocortical circuitry.8

Two candidate gene investigations of the one previously described functional SERT gene 5'-UTR variant, designated 5-HTTLPR, found preferential transmission of the L allele and an excess of the LL genotype in OCD.3,9 However, other studies of 5-HTTLPR in OCD reported no differences.10 One view of this conflicting literature follows from data indicating that OCD clinical heterogeneity may likely be obscuring possible associations.10,11

We sought to further investigate the SERT gene in OCD and two other serotonergic neuropsychiatric disorders by scanning its coding sequence using single strand conformational polymorphism (SSCP) analysis. We analyzed DNA samples from 170 unrelated individuals, including patients with OCD (n=30), eating disorder (n=30), and seasonal affective disorder (n=30) plus healthy controls (n=80).

Two uncommon variants were detected. An A to G transversion at nucleotide 1273 of the coding sequence, substituting Val425 for Ile425, was observed in two patients with OCD. Ile425 is conserved across all five mammalian species investigated, as well as the fly.12 The other variant, G1317A, was a synonymous substitution seen in one control. Neither of these uncommon SERT gene variants was observed in 213 additional unrelated individuals consisting of 143 OCD, eating disorder and seasonal affective disorder patients including 82 with OCD or a dual diagnosis of OCD and eating disorder plus 70 Centre d'Etude du Polymorphism Humain (CEPH) caucasian population controls.

We psychiatrically interviewed available relatives (A2 to 4, B2 to 10, Figure 1) of both OCD probands (A1 and B1) with Val425, and genotyped them for both I425V and 5-HTTLPR.13 Including the probands, seven of 14 members of these two pedigrees were heterozygous for 425V (Figure 1). Six of these seven individuals had OCD (n=5) or OC personality disorder (n=1). None of the other members of these families whom we studied had OCD or OC personality disorder. As summarized (Figure 1), the OCD probands and their sibs in the two families had an unusually severe clinical course with multiple and somewhat unusual coexistent neuropsychiatric disorders.

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Families with OCD and the I425V mutation. Two families with OCD probands with the I425V mutation and with other disorders: AS, Asperger's syndrome; MDD, major depressive disorder, Alc, alcohol abuse/dependence; AN, anorexia nervosa; OCPD, obsessive-compulsive personality disorder; SoP, social phobia; SiP, simple phobia; TicD, tic disorder; PTSD, Post-Traumatic Stress Disorder; ADHD, attention-deficit hyperactivity disorder. ?—Declined assessment interviews. Thus, six out of the seven individuals with I425V had OCD (n=5) or OCPD (n=6), while one individual who was diagnostically indeterminate was considered as non-OCD (Fisher's exact test two-sided P=0.005; relative risk=8.00; odds ratio=65.00).

Full figure and legend (119K)

Thus, this SERT gene mutation, which leads to defective regulation of SERT,14 may contribute to an uncommon familial form of OCD that is associated with Asperger's syndrome (AS)/autism, anorexia and a dense constellation of other serotonergic neuropsychiatric disorders proposed to be related to OCD. Owing of its rarity, I425V is not implicable in most individuals with these disorders. However, we have already observed the variant in two unrelated families, and I425V was also reported once in a SNP survey of 450 psychiatrically uncharacterized individuals.15 Therefore, it is likely that I425V, while uncommon, will be found in other families with OCD and related disorders.

Overall, OCD is a moderately heritable disorder which is cotransmitted in certain families with other disorders including OC personality disorder, depression, tic disorder and grooming disorders.7,16,17 Similar to the proposed action of 5-HTTLPR, I425V may confer an overall greater vulnerability to multiple disorders related to serotonin. However, the striking number, additional variety, severity and frequent treatment resistance of other disorders in the A and B families suggest that the actual phenotype defined by I425V is an atypical 'OCD plus': a specific syndrome incorporating OCD and an AS/autism/social phobia and eating disorder complex.

In fact, specific social difficulties were present in five of seven individuals with Val425, with two meeting criteria for AS and three for social phobia (SP). The AS found in two brothers is an unusual comorbidity since AS and autism are uncommon disorders with a population frequency of 2–15 in 10 000, vs 1–3% for OCD.18 Major studies of OCD comorbidity in adults do not mention AS/autism, while social phobia was found in only 1% of 1078 individuals with epidemiologically surveyed OCD,19 although some small studies found a higher incidence.16 AS is considered a pervasive developmental disorder related to autism but distinguished by normal language development and later onset with higher overall functioning.19 While repetitive behaviors are a superficial commonality between OCD and AS/autism, the interpersonal, cognitive, language and articulation deficits that are not seen in OCD, render these very distinct diagnoses.9,19,20

Family members of autistic probands have significantly higher rates of autism/AS (four- to nine-fold) plus SP, depression and alcohol/substance abuse and some particular personality traits, but not 15 other disorders, including OCD,21,22—except in one report.23 SP has 10-fold higher frequency (20%) in family members of autistic probands vs controls, and is observed most often in families of higher functioning autistic individuals without mental retardation (38% incidence), who are part of the broader autism spectrum.21,22 SP is characterized by fear and avoidance of interpersonal situations. Twin and family studies show SP to be hereditable,22,24 but, as with autism and OCD, contributory genes have not been found and verified. It is thus possible that these I425V families are linked, in addition to OCD, by AS/autism and SP, possibly representing a dimensional social dysfunction phenotype.

With regard to the multiple other disorders in these families, anorexia nervosa, found in two siblings with I425V, occurs in 5–13% of individuals with OCD and in 1% of the general population.25,26 Patients with anorexia nervosa frequently have OCD characteristics including perfectionism and compulsions related to both food and exercise. These observations might indicate that several disorders partially overlap in clinical comorbidity and in causality, including genetic transmission. I425V may operate as an aggravating factor for severity of these disorders.

These two probands and their families constituted the only individuals among >380 sampled who possessed the uncommon I425V mutation in the coding region of the SERT gene. One study employing complete sequencing of the SERT gene using DNA from 22 individuals with OCD plus controls revealed no variants.27 A recent report screening for low frequency of SERT gene sequence variations using SNPs found that the I425V mutation was rare, occurring in only one of the 450 individuals;14 no clinical or demographic data are available on these individuals.28

The I425V mutation is located in transmembrane region 8 of SERT (Figure 2), where it is in position to alter the secondary structure of this hydrophobic domain. Its presence locks the mutant transporter in an activated state that is normally achieved in the I425 transporter through the action of nitric oxide and cGMP. Evidence for constitutive activation of SERT by the 425V mutation in transfected human cell lines is presented elsewhere.14 Interestingly, in the two families we have studied, the I425V variant is on the same DNA strand with the L allele of 5-HTTLPR, a locus found at a distance of >2 kb from I425V. Furthermore, the homozygous 5-HTTLPR LL genotype is present both in the brothers A1 and A2 who have OCD plus AS and in the sisters B1 and B2 who have OCD or OC personality disorder plus AN. (Figure 1). The LL variant leads to two- to three-fold greater transcriptional activity (increased mRNA, SERT sites and serotonin uptake) plus greater response to cyclic AMP and protein kinase C than the SS form.13 Thus, this combination of SERT genotypes represents a unique 'double-hit' with functional consequences in the same, greater transport direction derived from both the coding region I425 mutation and the LL promoter region variant. The combined locked-on, gain-of-function effects of these SERT gene loci may cause an attendant deficit in the potential for adaptive responses to environmental challenges occurring either during early development or in adulthood.

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Topological location of the I425V mutation in transmembrane region 8 of the human serotonin transporter.

Full figure and legend (148K)

In conclusion, we have found an unusual clustering of multiple, uncommon neuropsychiatric disorders (0.02–2% of the general population for OCD, AN, AS/autism)2,18,25 associated with an uncommon functional missense variant in the serotonin transporter gene. Individuals with Val425 in two families had neuropsychiatric disorders long hypothesized to have a serotonergic etiological component. The unusual severity and treatment resistance of the disorders in the probands and their siblings likely represent the potent gain-of-function effect of Val425 on serotonin transporter function and may also reflect the combined 'double-hit' effect of Val425 with the more highly transcribed allele of the serotonin promoter polymorphism.




All patients and healthy controls gave informed consent prior to participating in human research protocols approved by the NIMH-IRP and the University of Pittsburgh School of Medicine Institutional Review Boards. Subjects were psychiatrically evaluated using the Structured Clinical Interview (SCID) for DSM–III–R and DSM-IV and/or Schedule for Affective Disorders and Schizophrenia (SADS) inventory prior to genotyping, as part of ongoing genetic studies of these disorders3,29,30,31 (except the CEPH controls and exceptions noted). Healthy controls had no history of any DSM Axis I psychiatric conditions.

SSCP-PCR, detection of the amino acid substitution by modified PCR and 5-HTTLPR genotyping

SSCP-PCR analysis was carried out according to procedures previously described,31 using 13 primer pairs that cover the entire SERT gene coding sequence (available from authors). A modified primer (5'GAT TAA CAT CAG AAA GAA GAT GC3') and the upper primer of exon 9 were used to introduce a base substitution (T to C) adjacent to the codon of interest, creating an artificial restriction site with only one allelic form.32 Following amplification of DNA, the PCR product was digested by HpaII yielding two fragments in the case of the rare allele Val425; the product remains intact in the case of the common allele, Ile425. All members of Family A and B were also genotyped for 5-HTTLPR using a modification of the genotyping procedure as previously described.13



  1. Ramamoorthy S, Bauman AL, Moore KR, Han H, Yang-Feng T, Chang AS et al. Antidepressant- and cocaine-sensitive human serotonin transporter: molecular cloning, expression, and chromosomal localization. Proc Natl Acad Sci USA 1993; 90: 2542–2546. | Article | PubMed | ChemPort |
  2. Lesch K-P, Wolozin BL, Estler HC, Murphy DL, Riederer P. Isolation of a cDNA encoding the human brain serotonin transporter. J Neural Transm 1993; 91: 67–72.
  3. Gelernter J, Pakstis AJ, Kidd KK. Linkage mapping of serotonin transporter protein gene SLC6A4 on chromosome 17. Hum Genet 1995; 95: 677–680. | PubMed |
  4. Androutsellis-Theotokis A, Rudnick G. Accessibility and conformational coupling in serotonin transporter predicted internal domains. J Neurosci 2002; 22: 8370–8378. | PubMed | ISI | ChemPort |
  5. Murphy DL, Li Q, Engel S, Wichems C, Andrews A, Lesch KP et al. Genetic perspectives on the serotonin transporter. Brain Res Bull 2001; 56: 487–494. | Article | PubMed | ISI | ChemPort |
  6. Lopez AD, Murray CC. The global burden of disease, 1990–2020. Nat Med 1998; 4: 1241–1243. | Article | PubMed | ISI | ChemPort |
  7. Nestadt G, Samuels J, Riddle M, Bienvenu OJ, Liang KY, LaBuda M et al. A family study of obsessive-compulsive disorder. Arch Gen Psychiatry 2000; 57: 358–363. | Article | PubMed | ISI | ChemPort |
  8. Bengel D, Greenberg BD, Corá-Locatelli G, Altemus M, Heils A, Li Q et al. Association of the serotonin transporter promoter regulatory region and obsessive-compulsive disorder. Mol Psychiatry 1999; 4: 463–466. | Article | PubMed | ISI | ChemPort |
  9. McDougle CJ, Epperson CN, Price LH, Gelernter J. Evidence for linkage disequilibrium between serotonin transporter protein gene (SLC6A4) and obsessive compulsive disorder. Mol Psychiatry 1998; 3: 270–273. | Article | PubMed | ChemPort |
  10. Cavallini MC, Di Bella D, Siliprandi F, Malchiodi F, Bellodi L. Exploratory factor analysis of obsessive-compulsive patients and association with 5-HTTLPR polymorphism. Am J Med Genet 2002; 114: 347–353. | Article | PubMed | ISI |
  11. Alsobrook JP, Leckman JF, Goodman WK, Rasmussen SA, Pauls DL. Segregation analysis of obsessive-compulsive disorder using symptom-based factor scores. Am J Hum Genet 1999; 88: 669.
  12. Demchyshyn LL, Pristupa ZB, Sugamori KS, Barker EL, Blakely RD, Wolfgang WJ et al. Cloning, expression, and localization of a chloride-facilitated, cocaine-sensitive serotonin transporter from Drosophila melanogaster. Proc Natl Acad Sci USA 1994; 91: 5158–5162. | Article | PubMed | ChemPort |
  13. Lesch K-P, Bengel D, Heils A, Sabol SZ, Greenberg BD, Petri S et al. Association of anxiety-related traits with a polymorphism in the serotonin transporter gene regulatory region. Science 1996; 274: 1527–1531. | Article | PubMed | ISI | ChemPort |
  14. Killic F, Murphy DL, Rudnick G. A human serotonin transporter mutation causes constitutive activation of transport activity. Mol Pharmacol, in press.
  15. Glatt CE, DeYoung JA, Delgado S, Service SK, Giacomini KM, Edwards RH et al. Screening a large reference sample to identify very low frequency sequence variants: comparisons between two genes. Nat Genet 2001; 27: 435–438. | Article | PubMed | ISI | ChemPort |
  16. Bienvenu OJ, Samuels JF, Riddle MA, Hoehn-Saric R, Liang KY, Cullen BA et al. The relationship of obsessive-compulsive disorder to possible spectrum disorders: results from a family study. Biol Psychiatry 2000; 48: 287–293. | Article | PubMed | ChemPort |
  17. Pauls DL, Alsobrook II JP, Goodman W, Rasmussen S, Leckman JF. A family study of obsessive-compulsive disorder. Am J Psychiatry 1995; 152: 76–84. | PubMed | ISI | ChemPort |
  18. Fombonne E. What is the prevalence of Asperger disorder? J Autism Dev Disord 2001; 31: 363–364. | Article | PubMed |
  19. McDougle CJ, Kresch LE, Goodman WK, Naylor ST, Volkmar FR, Cohen DJ et al. A case-controlled study of repetitive thoughts and behavior in adults with autistic disorder and obsessive-compulsive disorder. Am J Psychiatry 1995; 152: 772–777. | PubMed |
  20. Folstein SE, Rosen-Sheidley B. Genetics of autism: complex aetiology for a heterogeneous disorder. Nat Rev Genet 2001; 2: 943–955. | Article | PubMed | ISI | ChemPort |
  21. Piven J, Palmer P. Psychiatric disorder and the broad autism phenotype: evidence from a family study of multiple-incidence autism families. Am J Psychiatry 1999; 156: 557–563. | PubMed |
  22. Smalley SL, McCracken J, Tanguay P. Autism, affective disorder, and social phobia. Am J Med Genet 1995; 60: 19–26. | Article | PubMed | ISI | ChemPort |
  23. Bolton PF, Pickles A, Murphy M, Rutter M. Autism, affective and other psychiatric disorders: patterns of familial aggregation. Psychol Med 1998; 28: 385–395. | Article | PubMed | ISI | ChemPort |
  24. Kendler KS, Neale MC, Kessler RC, Heath AC, Eaves LJ. The genetic epidemiology of phobias in women. The interrelationship of agoraphobia, social phobia, situational phobia, and simple phobia. Arch Gen Psychiatry 1992; 49: 273–281. | PubMed | ISI | ChemPort |
  25. Fireman B, Koran LM, Leventhal JL, Jacobson A. The prevalence of clinically recognized obsessive-compulsive disorder in a large health maintenance organization. Am J Psychiatry 2001; 158: 1904–1910. | PubMed |
  26. Gorwood P, Bouvard M, Mouren-Simeoni MC, Kipman A, Ades J. Genetics and anorexia nervosa: a review of candidate genes. Psychiatr Genet 1998; 8: 1–12. | PubMed |
  27. Altemus M, Murphy DL, Greenberg B, Lesch KP. Intact coding region of the serotonin transporter gene in obsessive-compulsive disorder. Am J Med Genet 1996; 67: 409–411. | Article | PubMed |
  28. Collins FS, Brooks LD, Chakravarti A. A DNA polymorphism discovery resource for research on human genetic variation. Genome Res 1998; 8: 1229–1231. | PubMed | ISI | ChemPort |
  29. Enoch MA, Greenberg BD, Murphy DL, Goldman D. Sexually dimorphic relationship of a 5-HT2A promoter polymorphism with obsessive-compulsive disorder. Biol Psychiatry 2001; 49: 385–388. | Article | PubMed | ISI | ChemPort |
  30. Kaye WH, Lilenfeld LR, Berrettini WH, Strober M, Devlin B, Klump KL et al. A search for susceptibility loci for anorexia nervosa: methods and sample description. Biol Psychiatry 2000; 47: 794–803. | Article | PubMed |
  31. Ozaki N, Rosenthal NE, Pesonen U, Lappalainen J, Feldman-Naim S, Schwartz PJ et al. Two naturally occurring amino acid substitutions of the 5-HT2A receptor: similar prevalence in patients with seasonal affective disorder and controls. Biol Psychiatry 1996; 40: 1267–1272. | Article | PubMed | ISI | ChemPort |
  32. Haliassos A, Chomel JC, Grandjouan S, Kruh J, Kaplan JC, Kitzis A. Detection of minority point mutations by modified PCR technique: a new approach for a sensitive diagnosis of tumor-progression markers. Nucleic Acids Res 1989; 17: 8093–8099. | PubMed | ISI | ChemPort |


We thank our patient and family collaborators, Drs Gabriella Cora-Locatelli, Margaret Altemus, Theresa Pigott, Catherine Stayer, Joshua Rosenthal plus Diane Kazuba, Lucy Justement, Su-Jan Huang, Liz Maxwell, Julie Guroff, Teresa Tolliver and Theresa B DeGuzman for their contributions to this study, and Drs Klaus-Peter Lesch, Judith Rapoport and Sevilla Detera-Wadleigh for helpful suggestions with the manuscript. During the early stages of this work, Dr Norio Ozaki was supported at the NIAAA and NIMH Intramural Programs by Research Grants (10670923, 13470198, 13877152) from the Ministry of Science Education and Culture of Japan, the Research Grant for Nervous and Mental Disorders from the Ministry of Health and Welfare of Japan, and Special Coordination Funds for Promoting Science and Technology Target-oriented Brain Science Research Program from the Ministry of Science and Technology of Japan.