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March 2001, Volume 6, Number 2, Pages 160-167
Table of contents    Previous  Article  Next   [PDF]
Original Research Article
Genomic organization of the SLC1A1/EAAC1 gene and mutation screening in early-onset obsessive-compulsive disorder
J Veenstra-VanderWeele1, S-J Kim1, D Gonen1, G L Hanna2, B L Leventhal1,3 and E H Cook Jr1,3

1Laboratory of Developmental Neuroscience, Child and Adolescent Psychiatry, Department of Psychiatry, University of Chicago, MC3077, 5841 S Maryland Avenue, Chicago, IL 60637, USA

2Department of Psychiatry, University of Michigan, 1500 E Medical Center Drive, Ann Arbor, MI 48109, USA

3Department of Pediatrics, University of Chicago, MC3077, 5841 S Maryland Avenue, Chicago, IL 60637, USA

Correspondence to: E H Cook Jr MD, University of Chicago, Department of Psychiatry MC3077, 5841 South Maryland Avenue, Chicago, IL 60637, USA. E-mail: ed@yoda.bsd.uchicago.edu

Abstract

The first genome scan conducted in early-onset obsessive-compulsive disorder used a non-parametric analysis to identify a peak in a region of chromosome 9 containing the gene SLC1A1, which codes for the neuronal and epithelial glutamate transporter EAAC1. Interaction between the glutamatergic and serotonergic systems within the striatum suggests EAAC1 as a functional candidate in OCD as well. We determined the genomic organization of SLC1A1 primarily by using primers designed from cDNA sequence to amplify from adaptor-ligated genomic DNA restriction fragments. In order to confirm SLC1A1 as a positional candidate in early-onset OCD, common single nucleotide polymorphisms (SNPs) were identified that enabled mapping of SLC1A1 within the region of the lod score peak. Based on the linkage evidence, the coding region was sequenced in the probands of the seven families included in the genome scan. No evidence was found for a functional mutation, but several SNPs were identified. Capillary electrophoresis SSCP typing of a haplotype consisting of two common SNPs within EAAC1 revealed no significant linkage disequilibrium. Molecular Psychiatry (2001) 6, 160-167.

Keywords

obsessive-compulsive disorder; glutamate; transporter; polymorphism

Introduction

The first genome scan for linkage in early-onset obsessive-compulsive disorder (OCD) used a non-parametric analysis to identify linkage to the 9p24 region that peaked at 9.03 cM from 9pter (NPL score 3.19, P = 0.0032).1 Despite the paucity of cytogenetic findings in this disorder, 9p monosomy has been reported in a single patient with Tourette's syndrome and OCD.2 The region in common between these two findings contains only one currently identified brain-expressed gene, SLC1A1.3 SLC1A1 codes for EAAC1, a neuronal and epithelial glutamate transporter gene in the same gene family as the other Na+-excitatory amino acid transporters GLT-1, GLAST, EAAT4, and EAAT5.4 While antisense oligo studies of the glutamate transporters suggest major roles for GLT-1 and GLAST in preventing neurotoxicity, EAAC1 appears to play a minor role.5 Mice lacking GLT-1 develop lethal spontaneous seizures and increased susceptibility to cortical injury,6 but mice lacking EAAC1 manifest only dicarboxylic aminoaciduria and reduced spontaneous motor activity.7

Glutamatergic neurotransmission has not been extensively studied in obsessive-compulsive disorder. One study found decreased glutamate resonance in the caudate after paroxetine treatment of a pediatric patient with OCD.8 Alterations in serotonergic neurotransmission following inhibition of the serotonin transporter are thought to underlie the therapeutic effects of serotonin reuptake inhibitors in OCD.9 NMDA and AMPA/kainate glutamate receptors also appear to play a role in modulating the release and metabolism of serotonin in rat striatum,10 a portion of the brain implicated in OCD.11,12

Two lines of evidence support SLC1A1/EAAC1 as a candidate gene in OCD. SLC1A1 is a positional candidate on the basis of its proximity to the non-parametric linkage peak on chromosome 9. Additionally, based on the documented interaction between serotonergic and glutamatergic systems in the caudate nucleus and limited findings relating glutamate function to OCD, EAAC1 is also a functional candidate in OCD. In order to screen SLC1A1 for mutations, we first determined the genomic structure surrounding the known cDNA sequence.13 Then, since SLC1A1 is a stronger positional than functional candidate gene, we sought to identify polymorphisms that would confirm placement of the gene relative to the lod score peak. Once SLC1A1 was mapped within the region of interest, we screened the coding region for mutations using exon-flanking primers in one affected relative from each of the seven extended pedigrees. We used two of the identified polymorphisms to evaluate possible linkage disequilibrium between the gene and early-onset OCD, both within the original linkage families and within an extended sample including primarily simple parent-child trios.

Materials and methods

Genomic organization of SLC1A1

While the genomic structure of SLC1A1 was not previously studied in any species, the organization of another glutamate transporter gene SLC1A2 (also known in the literature as GLT-1 and EAAT2) was previously elucidated in humans.14 Using an alignment of the amino acid sequences of each transporter,4 it was possible to predict the boundaries for some of the exons of SLC1A1. Unidirectional nested oligonucleotide primer sets were designed according to these predictions. These primer sets were used along with a nested set of adaptor primers to amplify from adaptor-ligated genomic DNA restriction fragments (GenomeWalkerÔ, Clontech, Palo Alto, CA, USA). Introns 1, 3, 4, 6, 7, and 8 were not as predicted by the SLC1A2 genomic structure, so new nested oligonucleotide primer sets were designed. Exon 5 was only 40-bp long, making it impossible to design nested primer sets within it. Oligonucleotide primers were used to sequence flanking sequence in both directions out of a PCR product that spanned introns 4 and 5. The GC content of exon 1 and immediately flanking sequence proved too high (73%) for amplification using Tth polymerase with and without GC-melt reagent. However, substituting the DyNAzymeÔ EXT DNA polymerase (Finnzymes Oy, Espoo, Finland) enzyme system along with 1.5 M GC-melt reagent (Clontech) enabled amplification from the GenomeWalkerÔ restriction fragment libraries. In order to fill in sequence surrounding exons 1, 8 and 9, the Human BAC Personal Size DNA Pools (Research Genetics, Huntsville, AL, USA) was used to identify BAC clones (269c 13, 432a14, 457h8) that contained an amplicon in intron 1. These clones were then used to amplify regions that could not be amplified from the GenomeWalkerÔ restriction fragment libraries. PCR products were sized on 1% agarose gels, and excised fragments were extracted using the QIAquick Gel Extraction Kit (Qiagen, Valencia, CA, USA). Cycle sequencing was performed using the ABI prism dRhodamine Terminator Cycle Sequencing Ready Reaction Mix or, in the case of exons 1, 8, and 9, the ABI prism dGTP BigDyeÔ Terminator Cycle Sequencing Ready Reaction Mix (PE Applied Biosystems, Foster City, CA, USA). Sequencing fragments were separated by capillary electrophoresis on an ABI prism 310 Genetic Analyzer (PE Applied Biosystems).

Intron sizes were determined by pairing the unidirectional nested primer sets described above and amplifying with the Advantage Genomic Polymerase Kit (Clontech) from a pool of 10 unrelated control subjects, or if that amplification failed, from the BAC clones listed above. Using this method, a control fragment of 18.7 kb was amplified successfully. Introns 1 and 2 could not be amplified using either of these methods. Intron size was estimated from separation on a 1% agarose gel and is given in Table 1.

Identification of common polymorphisms

Primers were designed to amplify each exon and at least 50 bp of flanking intron sequence. These primer sets, given in Table 2 below, were used to amplify a genomic DNA pool of 10 unrelated control subjects. Except for exon 1, amplification was carried out using the AmpliTaq Gold DNA Polymerase system (PE Applied Biosystems). For amplification of exon 1, we used two separate primer sets, as given in Table 1, with the DyNAzymeÔ EXT DNA polymerase (Finnzymes Oy) enzyme system. PCR products were sized on 4% agarose gels, and excised fragments were extracted using the QIAquick Gel Extraction Kit (Qiagen). Cycle sequencing was performed in one direction using the ABI prism dRhodamine Terminator Cycle Sequencing Ready Reaction Mix (PE Applied Biosystems). Sequencing fragments were separated by capillary electrophoresis on an ABI prism 310 Genetic Analyzer (PE Applied Biosystems).

Subject ascertainment and diagnosis

The probands in the genome scan were five boys and two girls with a current diagnosis of definite OCD. They ranged in age from 6 to 17 years (14.4 ± 3.8 years). OCD onset age in the probands ranged from 3 to 14 years (8.8 ± 3.9 years). Three probands also had a history of Tourette's disorder. The probands were recruited from clinics at the University of Michigan Medical Center and local chapters of the Obsessive-Compulsive Foundation as part of a family study of early-onset OCD. Each proband was directly interviewed to determine whether they met DSM-III-R criteria for OCD.15 Exclusion criteria for the probands were: (1) chronic neurological disorder (other than tic disorder); (2) mental retardation; (3) DSM-III-R diagnosis of autistic disorder, schizophrenia, or bipolar disorder; (4) currently living away from both biological parents; and (5) adoption. Written informed consent was obtained from both parents and assent was obtained from each proband. The study was approved by the Institutional Review Board of the University of Michigan Medical Center.

After completing the proband diagnostic evaluation, permission to contact other relatives was requested from the parents. Direct interviews were completed with all 26 first-degree relatives. Direct interviews were completed with 20 of 22 second-degree relatives (91%) contacted for further diagnostic evaluation; however, a blood sample was not obtained from one interviewed second-degree relative. Direct interviews were completed with 13 more distant relatives in two families.

A genome scan was completed with 65 individuals from seven families with two or more affected relatives. The 28 affected relatives consisted of 25 individuals with definite OCD and three individuals with subthreshold OCD. Six affected relatives were siblings, four were parents, nine were second-degree relatives, and nine were more distant relatives. They ranged in age from 12 to 89 years (42.7 ± 20.3 years). OCD onset age in the affected relatives ranged from 5 to 24 years (12.6 ± 5.6 years). Two of the affected relatives also had a history of chronic tics. The 29 unaffected relatives ranged in age from 12 to 80 years (44.5 ± 19.8 years). None of the unaffected relatives had a tic history. A 10-year-old girl with possible compulsions was considered unknown. An additional 21 parent-child trios and two parent-child pairs containing probands and parents with no other affected siblings or second-degree relatives were included in linkage disequilibrium studies.

After informed consent and assent were obtained, all probands and all siblings between 10 and 17 years were interviewed with the Schedule for Affective Disorders and Schizophrenia for School Age Children-Epidemiologic Version (K-SADS-E).16 Siblings and other relatives less than 10 years were not included in the study. The interview was completed independently with a parent of the subject as well as with the subject. The interview was supplemented with the sections on OCD and the tic disorders from the Schedule for Tourette and Other Behavioral Syndromes (Version C1).17 Relatives 18 years and older were interviewed with the Structured Clinical Interview For DSM-III-R (SCID)18,19,20 and the sections on OCD and the tic disorders from the Schedule for Tourette and Other Behavioral Disorders (Version A1).21

Both interviews included a version of the Yale-Brown Obsessive Compulsive Scale (Y-BOCS22,23) modified to obtain information about the lifetime occurrence of obsessive and compulsive symptoms. A series of screening questions designed to cover all criteria for a DSM-III-R diagnosis of OCD preceded the Y-BOCS checklist.24,25 All other parts of the interview were identical to either the K-SADS-E (child) or the SCID (adult). The same interviews were used in the diagnostic assessment of all first-, second-, and third-degree relatives included in the genome scan.

Additional information on relatives 18 years and older was obtained with the Family Informant Schedule and Criteria (FISC).26 The mother of each affected offspring was interviewed with the FISC regarding her spouse, adult offspring, parents, and siblings. The father of each affected offspring was interviewed with the FISC regarding his spouse, parents, and siblings. The maternal grandmother provided FISC information for the third-degree relatives in the largest pedigree (see Figure 1). Thus, two types of data were obtained on all adult subjects: (1) information from direct structured interviews; and (2) personal history information from a biological relative and/or spouse.

All interviews were audiotaped as well as coded on paper. Subjects who had received psychiatric or neurological services were asked to sign a written release of information form so that copies of their clinical records could be requested and added to the structured interview data.

All interviewers had at least a master's degree and clinical training in child or adult psychopathology. They were trained to at least 90% diagnostic agreement with the individual instruments. The interviewers were confined to interviewing either probands and siblings between 10 and 17 years of age or adult relatives. The interviewer for a given proband was not involved with the interviews of other family members. Because normal control probands and their relatives were included in our ongoing family study, the interviewers were blind to proband status.

After completion of all interviews for an individual, all available materials (personal interview data, family history data, and clinical records) were collated. All information identifying or describing the proband was removed so that diagnostic ratings could be completed by raters blind to proband diagnosis. The blind diagnosticians were never given a complete family to evaluate at one time, and all diagnostic evaluations of probands were done separately from those of the relatives.

Best-estimate diagnoses were made independently by two investigators using DSM-III-R criteria. If a subject had sufficient symptoms to meet all criteria, a 'definite' diagnosis was assigned. A 'subthreshold' diagnosis was made if a subject definitely had a history of obsessions and/or compulsions, but lacked compelling evidence for the following criterion: (1) marked distress; (2) duration of obsessive-compulsive symptoms for more than one hour a day; or (3) significant interference in the person's normal routine, occupational (or academic) functioning, or usual social activities or relationships with others. When major disagreements occurred between two diagnosticians, consensus diagnoses were reached with the assistance of a third diagnostician following established procedures developed for the diagnosis of other psychiatric disorders.27,28

Relatives with a lifetime diagnosis of definite OCD were considered affected in a narrow phenotype definition. Relatives with a lifetime diagnosis of either subthreshold or definite OCD were considered affected in a broad phenotype definition. Relatives with a history only of probable obsessions or compulsions were considered unknown. Relatives with a lifetime diagnosis of tic disorder or trichotillomania, but without a lifetime diagnosis of either definite or subthreshold OCD, were also considered unknown.

Blood collection and DNA extraction

Peripheral blood samples were obtained by venipuncture from consenting subjects. Saliva samples were obtained from those who refused venipuncture. Samples were immediately transported to the laboratory and stored frozen at -70°C until the time of DNA extraction. DNA was extracted using the PureGene DNA Isolation Kit (Gentra Systems, Minneapolis, MN, USA).

Genotyping and recombination analysis

Two polymorphisms were used to confirm the location of SLC1A1 within the lod score peak on chromosome 9. These two single nucleotide polymorphisms (SNPs) were amplified in one PCR product with an unlabeled sense primer (SLC1A1 1A 5'-gcccattgtctgtagatgagaac-3') and a fluorescently labeled antisense primer (SLC1A1 1B 5'-tggtacagaaataatacacgacgac-3') using AmpliTaq Gold DNA Polymerase (see conditions given above) for 31 cycles at an annealing temperature of 55.2°C. Genotypes were distinguished by single-stranded conformational polymorphism (SSCP) analysis on the ABI 310 Genetic Analyzer using SNAP polymer (PE Applied Biosystems) and Genescan TAMRA 350 (PE Applied Biosystems) as an internal size standard.29 Sixteen published markers (see Table 2) on distal 9p at an average distance of 1 cM were also amplified in a set of 56 subjects from the seven families used in the childhood-onset OCD genome screen. Estimated recombinations between these markers were calculated using GENEHUNTER software.30

Genotyping and linkage disequilibrium analysis

Linkage disequilibrium was initially assessed using only subjects contained within the seven linkage families collected as above. Each affected person within a pedigree was considered as a separate proband for the generation of 19 parent-child trios and five parent-child pairs. This sample was later extended by the addition of 21 parent-child trios and two parent-child pairs containing probands and parents with no other affected siblings or second-degree relatives.

The two polymorphisms amplified within the SLC1A1 1A/1B PCR product as above were also used to evaluate linkage disequilibrium. Most genotypes were determined by SSCP analysis on the ABI 310 Genetic Analyzer as above. However, 18 genotypes were determined by cycle sequencing, performed as above using SLC1A1 1A/1B. Sequencing fragments were separated by capillary electrophoresis on an ABI prism 310 Genetic Analyzer (PE Applied Biosystems). Haplotypes within these 18 subjects were determined based on haplotype combinations directly observed in transmission patterns. Haplotypes were compatible with Mendelian transmission of haplotypes and individual alleles in each family. Data were analyzed by the multiallelic TDT (MTDT).31,32

Mutation screen in childhood-onset OCD probands

DNA was extracted from the blood of six of the seven probands seen at the University of Michigan as described above.1 Due to insufficient blood from one of the seven probands, an affected first-degree relative of the proband was substituted. PCR products were amplified and sequenced in one direction as described above. For exon 12 and in other instances of insufficient clarity or read length in one direction, subjects were sequenced in the other direction as well. Figure 2 shows a typical sequencing electropherogram.

Results

Genomic organization of the SLC1A1 gene

Exon-intron junctions for SLC1A1 are given in Table 1. The genomic organization of SLC1A1 is similar to that of three other glutamate transporter genes SLC1A2, SLC1A3, and SLC1A6. Five exon-intron junctions are shared among all four genes in the human. An additional three exon-intron junctions are shared by SLC1A1, SLC1A3 and SLC1A6. SLC1A1 contains an additional three introns shared with none of the other glutamate transporter genes. Flanking sequence for each exon of SLC1A1 has been deposited with the EMBL/GenBank Data Libraries under Accession Numbers AF074903-AF074911, AF143773. Primer sets to amplify each exon are given in Table 2.

Mutation screen, SNP identification, polymorphism typing and mapping by analysis of recombinations

All seven subjects were sequenced in at least one direction for each of the 12 primer sets covering the 11 exons in the SLC1A1 coding region. Eight single nucleotide polymorphisms (SNPs) were identified within either exons or flanking intronic regions of SLC1A1. An A to T transversion that did not affect predicted splice sites was identified 12 bp from the 5' end of intron 2 at base 462 of the exon 2 genomic sequence. Two transitions were identified 72 and 25 bp, respectively, from the 3' end of intron 2: C to T at base 224, and C to T at base 271 of the exon 3 genomic sequence. Two transitions were identified 52 and 107 bp, respectively, from the 5' end of intron 3: G to A at base 446, and G to A at base 501 of the exon 3 genomic sequence. A G to A transition that did not change the amino acid sequence was identified within exon 4 at base 301 of the exon 4 genomic sequence. A G to A transition that did not affect predicted splice sites was identified 8 bp from the 5' end of intron 8 at base 508 of the exon 8, 9, and 10 genomic sequence. A C to T transition that did not change the amino acid sequence was identified within exon 9 at base 509 of the exon 9 genomic sequence.

Two of these SNPs, at base 224 and 271 within the exon 3 genomic sequence, were genotyped in 56 subjects. The transition at base 224 of the exon 3 genomic sequence had an observed heterozygosity of 0.54 (38/70 parents of probands), and the transition at base 271 had an observed heterozygosity of 0.26. There were only six haplotype combinations between the two polymorphisms (see Figure 3), as the C allele at base 271 always co-occurred with the T allele at base 224. Eighteen total subjects were sequenced to confirm the haplotypes. The resulting haplotypes led to no incompatibilities within the 56 subjects in seven families, nor within the additional families added later for linkage disequilibrium analysis. The observed heterozygosity for the haplotypes of the two polymorphisms was 0.56 (40/70 parents of trios or pairs). Recombinations between the 16 markers on distal 9p (see Table 3) were consistent with a placement of these two SLC1A1 polymorphisms between D9S199 and D9S1852.

Linkage disequilibrium analysis

No significant evidence of linkage disequilibrium was found between early onset OCD and SLC1A1 either in the families with more than one affected relative (MTDT 3.41, 2 df, P = 0.18) or all families (MTDT 1.71, 2 df, P = 0.42) (Table 4).

Discussion

Mapping common single-nucleotide polymorphisms within SLC1A1 relative to the lod score peak confirmed that SLC1A1 is a positional candidate gene for childhood-onset OCD based on a previous genome screen. Screening of the coding region in the probands of the seven families involved in the genome scan revealed no mutations that would be likely to be responsible for disrupting expression or activity of the SLC1A1 gene product. Given that screening was unidirectional, it must be noted that a mutation may have been missed.

Several SNPs were identified in SLC1A1. Two adjacent SNPs were typed by CE-SSCP and the haplotype patterns were called and found to be consistent with sequencing and were compatible in the families. Significant linkage disequilibrium was not found between this locus and early-onset OCD. However, the sample size is relatively small and only one part of the gene was screened. Genotyping of other SNPs for SLC1A1 will be necessary to fully test the possibility of linkage disequilibrium between a part of the gene and early-onset OCD.

The SNPs reported here will enable other investigators to use linkage disequilibrium methods to assess the role of SLC1A1 in other psychiatric or neurologic disorders. The genomic organization of SLC1A1 will allow mutation screening of this gene in other phenotypes as well.

A recent study reports a splicing variant of SLC1A1 that lacks exon 2, which corresponds to the first extracellular domain.33 After the regulation of this splicing variant and the function of the promoter region are clarified, analysis of the promoter region or other regulatory gene elements would be a logical next step in searching for possible mutations of this gene in childhood-onset obsessive-compulsive disorder. Additionally, given the absence of significant linkage disequilibrium, other genes in the region must be considered positional candidates. However, given that the linkage finding in the region does not meet levels of significance for definite linkage, the region may not contain a susceptibility locus.

Acknowledgements

Shuya Yan provided expert technical assistance. This work was supported, in part, by NIH K02 MH01389 (EHC), NIH K20 MH01065 (GLH), NIH R01 MH58376 (GLH), the Jean Young and Walden W Shaw Foundation (BLL), the Harris Foundation (BLL), the Brain Research Foundation (EHC), and the Obsessive Compulsive Foundation (EHC, GLH).

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Figures

Figure 1 This pedigree shows the largest family included in the genome screen.

Figure 2 This electropherogram shows 450 bp of sequence spanning exon 6 with flanking intron sequence.

Figure 3 Electropherograms are shown for the six patterns observed on SSCP between the two SLC1A1 polymorphisms. Length units assigned using Genescan TAMRA 350 as an internal size standard are displayed on the horizontal axis and fluorescent intensity is displayed on the vertical axis. Each genotype was confirmed by cycle sequencing. Six peak patterns were seen, corresponding to different combinations of the following three observed haplotypes: T-T, C-T, and T-C. The length units of each peak of these haplotypes are labeled on the three subjects that were homozygotes for these haplotypes. The C-T haplotype was distinguished from the T-T haplotype by a shift of the shorter migrating band right by two length units and a shift of the longer migrating band left by three length units. The T-C haplotype was distinguished from the T-T haplotype by a shift of the longer migrating band right by five length units. The C-C haplotype was not observed, but following the pattern of the other three haplotypes, it should be possible to distinguish on the basis of a short band two length units right of the T-T short band and a long band between the T-T long band and the T-C long band. In our population, the C allele at 271 only occurred in the presence of the T allele at 224. Haplotype analysis within 56 subjects from seven linkage families confirmed the absence of the C-C haplotype in cases where subjects were shown by sequencing or SSCP to be heterozygous at both loci. An absence of observed recombinations is reasonable for our sample since if a centimorgan is assumed to be approximately equal to a megabase, the two markers would be estimated to have a 5 ´ 10-7 chance of recombining per meiosis. Other populations may have a different distribution of haplotypes and should have haplotypes confirmed by sequencing and linkage analysis.

Tables

Table 1 Exon-intron junctions of the human SLC1A1 gene

Table 2 Oligonucleotides

Table 3 Estimated recombinations

Table 4 Transmission data for each haplotype of SLC1A1

Received 5 March 2000; revised 14 July 2000; accepted 14 July 2000
March 2001, Volume 6, Number 2, Pages 160-167
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