Molecular Psychiatry (2001) 6, 387–395  2001 Nature Publishing Group All rights reserved 1359-4184/01 $15.00 www.nature.com/mp ORIGINAL RESEARCH ARTICLE Mutation analysis of SYNJ1: a possible candidate for 21q22-linked bipolar disorder T Saito, F Guan, DF Papolos, S Lau, M Klein, CSJ Fann and HM Lachman

Department of Psychiatry, Division of Psychiatry Research, Albert Einstein College of Medicine, Bronx, New York 10461, USA

Genes involved in the regulation of synaptic vesicle function are potential candidates for the development of psychiatric disorders. In addition to experimental and theoretical consider- ations, a number of involved in synaptic vesicle function map to regions of the genome that have been linked to bipolar disorder (BPD) and schizophrenia (SZ). One is synaptojanin 1(SYNJ1) which maps to 21q22.2, a chromosomal region that has been linked to BPD in a subset of families in several studies. Synaptojanin 1 is an 5- that has an important role in synaptic vesicle endocytosis. Mutation screening of 32 exons, intron–exon junctions, and 839 bases of 5Ј-flanking DNA resulted in the identification of 11 mutations of which four were very common and seven were very rare. Of the 11 mutations identified, sev- eral may have functional significance including two coding variants, two that may affect the binding of a transcription factor, and two that involve known splicing regulatory domains. Five bipolar patients out of 149 analyzed were found who have one of the four rare variants that were most likely to have functional significance compared with 0/148 controls. The allele frequencies for three of the four common variants were very similar in bipolar patients and controls. A slight difference in allele frequency was found for an interesting mutation we detected in intron 12 in which two non-adjacent thymidine residues are deleted in a poly-AT tract located near the exon 12 splice donor site (␹2 = 2.45, P = 0.12, 2-tailed). Although we failed to unequivocally identify a specific SYNJ1 allele that could be responsible for putative chromosome 21q22-linked BPD, several interesting variants were found to be increased in bipolar subjects and should be further investigated. Molecular Psychiatry (2001) 6, 387–395. Keywords: SNP; SNARE; polymorphism; candidate gene; ; manic depression; manic depressive illness

Introduction cal features of ADHD overlap with those found in mania and hypomania. Fifth, altered expression of the Genes involved in the regulation of synaptic vesicle synaptic SNAP-25, synaptophysin, and com- function are good candidates to consider for BPD sus- plexins I and II, have been found in the brains of ceptibility. This idea is based on several observations. 5–9 + schizophrenics and bipolar individuals. Finally, a First, Ca2 and phosphoinositides, which are critical number of genes encoding homologs of SNARE and mediators of synaptic vesicle function, are targets for vesicle proteins, and their regulators, map to regions some of the drugs used to treat BPD.1 Second, of the genome that are suspected to harbor BPD and SZ depletion of vesicle catecholamines by reserpine may susceptibility genes. SNAP29, RAB36, phosphatidyl- cause depression, especially in patients with underly- inositol 4-kinase (Pl4K), synapsin III and synaptogyrin ing mood disorders.2 Third, amphetamine, which 1 map to chromosome 22q11–13,10–16 synapsin I and induces behavioral changes similar to those found in synaptophysin map to Xp11,17 SYBL1 maps to Xq28,18 SZ and in mania, causes the redistribution of cat- syntaxin 7 maps to 6q2119–21 and SYNJ1, which echolamines from vesicles to cytosol.3 Fourth, deletion encodes synaptojanin, a phosphatase enriched in pre- of the gene encoding the SNARE SNAP-25 in synaptic terminals, maps to 21q22.2.22–28 the coloboma mouse strain causes behavioral alter- Phosphoinositides are important regulators of vesicle ations that mimic those found in patients with atten- exocytosis and endocytosis.29 The endocytic proteins tion deficit hyperactivity disorder (ADHD).4 The clini- synaptojanin, 1 and amphiphysin, and synap- totagmin, a synaptic vesicle protein involved in exocytosis through its role as a Ca2+ sensor, all bind Correspondence: HM Lachman, Department of Psychiatry, phosphoinositides.30–34 Synaptojanin regulates endo- Division of Psychiatry Research, Albert Einstein College of Medi- cytosis by dephosphorylating soluble and lipidic phos- cine, 1300 Morris Park Ave, Bronx, New York 10461, USA. E- Ј mail: LachmanȰaecom.yu.edu phoinositides at the 5 position of the inositol ring. Its Received 4 October 2000; revised 11 December 2000; accepted 19 targets include phosphatidylinositol 4,5-bisphosphate December 2000 (Pl(4,5)P2) and phosphatidylinositol 3,4,5-trisphos- Mutation analysis of SYNJ1 T Saito et al

388 29–32 phate (Pl(3,4,5)P3. The of and AP000277). Primers were derived from intron Pl(4,5)P2 by synaptojanin is a critical step in the sequences and were designed to amplify exons and uncoating of from endocytic vesicles. intron–exon junctions. Overlapping primers were used The SYNJ1 gene encodes 170-kDa and 145-kDa syn- to amplify the 5Ј-flanking region. The primers used in aptojanin isoforms that are generated by alternative this study are shown in Table 1. The SYNJ1 gene is splicing; synaptojanin-170 is expressed at low concen- cloned in the opposite orientation in all of the genomic trations in many tissues and fetal brain whereas synap- clones in GenBank. However, the polymorphisms tojanin-145 is primarily found in nerve terminals.33–35 reported in this study are reported from the perspective The SYNJ1 gene encodes three distinct functional of the sense strand. domains. The central portion encodes the inositol 5- PCR-amplified genomic DNA fragments were phosphatase activity. The 5Ј-region is homologous to screened for polymorphisms using a modification of the cytosolic portion of the yeast Sac1 protein, which the single strand conformation polymorphism (SSCP) has been implicated in the regulation of vesicular traffic. The Sac1-like homology has been shown to have an intrinsic polyphosphoinositide phosphatase activity Table 1 Primers used to amplify SYNJ1 exons and intron separate from that encoded by the central 5-phospha- exon junctions tase domain.36,37 The 3Ј end encodes a proline-rich region that is involved in binding synaptojanin to Exon Forward primer Reverse primer Size amphiphysin and other proteins found on endocytic vesicles.33,34,37,38 1 ggcgcaatgcggaagagatg gacgctgcggaggcagagac 200 Based on its role in vesicle recycling, as well as its 2 atgctttgcagtccttgctg ggttgcatctgaattgtttcaa 283 location on 21q22.2, a region of the genome mapped 3 gaatccaatgtttcaggtca ctctcaaatccttagaattac 206 in a subset of families with BPD, we viewed SYNJ1 as 4 aggtgcaagaagaaatggaa actgaacaactgctgaacat 400 5 catactttgttacttagtat tgttattctagtattttctgtg 350 a credible candidate gene for BPD. 6 gtaagtgtaacaacttttct actatccagaaagtttagaa 210 7 gtgtgttcagcaaagaaatt gaagtgaatcagtaaataca 240 Methods 8 attgaaaaaattgggcttc aataccattctaggggtcca 210 9 ttagatggatatttgccttt atgttccaattcaaagaaca 270 Subjects 10 cctcaattattcactgtttg ttaaagaggcagatgataca 203 Patients with BPD were diagnosed on the basis of 11 tctggtagttcttaaaagcc ggccatttaaaagctttatgaag 299 either a SADS-L interview or by unstructured clinical 12 aaaacctaatgagtaaaatcag gtttacatttacctcttggcc 291 interview modified from SADS-L that yielded diagnos- 13 ttttgtcactatactcaacattt ccaattatatggctttatttgt 195 tic information enabling the diagnosis of BPD using 14 tttataaatggtttgatgcagc acacagtcaggatttattaac 290 RDC criteria.39,40 Patients with either bipolar disorder 15 gttggatggggttgctattt agtttccctcaactggcaaa 201 I (BPI) (n = 95) or bipolar disorder II (BPII) (n = 54) were 16 ttgtcataaaataggctattgt tttcttgttatttaatggtagg 263 17 tcttgaaatttcaaatctgcga tatatttcaaagctttatcttc 284 included in the study. The patients were recruited from 18 aagcatatctcaggcaaaga cgacccaaaatcttaatgca 291 clinics and private practices affiliated with the Albert 19 tgtctgaatgaaaacagaac tcataagtttacattagttcatt 272 Einstein College of Medicine. Forty-three subjects were 20 tcctgcttatacagcttctc caaacaatttcaaacactatta 219 unrelated patients with BPI obtained from the Coriell 21 gaaatttgtttgaaatacacc cccaaataaggtgatttcta 358 Institute in conjunction with the The National Insti- 22 gcttgttatgtctatggtat agataatcttgctgcatgtc 220 tutes of Mental Health Bipolar Genetics Initiative. 23 gtcagcctctttattccaaa cagattggaagaaaacatatc 292 Patients signed an informed consent approved by the 24 tggagttatctctgatcaag catcaagcacattggccaca 300 local Committee on Clinical Investigation. Control sub- 25 atcttcttacctcctgtgcg gagtgaagaattttacatctagc 220 jects were students, anonymous blood bank donors, 26 atactatagcaatctttgac ctttcaggtgtaaaacattaat 181 27 gtttactatggaaattgtct gggcgctgacactgagaaa 199 anonymous DNA samples obtained from liver biopsies 28/29 agcaagctgatgttatgcaa tagtagtaaacaggacttag 370 in consenting patients undergoing abdominal surgery, 30 agaattttacctgtggaagg aaattttctgggctcaagtgatc 220 and hospital, medical school, and clinic staff. No for- 31 cttccttcattataccacaa ggtatgttcaaagactaaatc 425 mal testing procedure was used to screen controls for 32 aaagcattttgttgactgaa ttcatacatctaatgtgttg 180 underlying psychiatric illness. All patients and con- trols were Caucasian. There were 79 female and 70 5Ј-flanking male controls (mean age 45.5 ± 15.2 female, 44.3 ± 15.7 male), and 83 female and 66 male patients with BPD pro1 gtagctggagactgaggctg ccgggagcgtcacttccgct 180 (mean age 40.4 ± 10.8 female, 41.0 ± 11.7 male). pro2 acgctctgaagagcccacgc ccacgcccactctgcgccga 180 pro3 acctggttggctcggcgagc gagggaagatctggcctcct 200 pro4 gacgtcccactctccatcgt ggaagggctcgggacggtcg 240 Mutation detection and genotyping pro5 gcagaaatctatgggacacg cgaagaatgcttaggtgtat 240 DNA was extracted from whole blood or buccal smears using a Puregene DNA isolation kit (Gentra Systems, PCR primers are derived from intron sequences and flank the Minneapolis, MN, USA) or by standard phenol extrac- exon number shown with 5Ј to 3Ј direction from left to right. tion methods. SYNJ1 exons and intron–exon junctions Restriction enyzmes were used for exon primer sets 4 (EcoR1), were mapped by comparing the cDNA sequence with 5(Cac81), 21 (Sau96l) and 31 (Bsu361) to reduce size of PCR chromosome 21 genomic DNA (GenBank accession product for SSCP. Size of PCR product in base pairs shown numbers AF009039, AP000181, AP000105, AP000182, at right.

Molecular Psychiatry Mutation analysis of SYNJ1 T Saito et al 389 analysis procedure originally described by Orita et al.41 the four common polymorphisms are shown in Forty-five DNA samples obtained from unrelated Figure 1a. Positive controls for each allele were patients with BPI were used in the screening. PCR was included on each gel used for genotyping. When there performed in a 20-␮l reaction volume containing 50 ng were discrepancies, the gels were reviewed together genomic DNA, a mixture of all four nucleotides (final and if no agreement could be made, the analysis was concentration 0.2 mM for each), 1 × reaction buffer, repeated. 20 pM of each primer and 1.25 U taq polymerase (Gibco-BRL, Grand Island, NY, USA). A list of all the Computer analysis primers used in this study and the size of the PCR pro- The BLAST network at the National Center for Biotech- duct are shown in Table 1. For mutation screening by nology Information was used to analyze DNA SSCP using a minigel system (Novex, Santa Clarita, CA, sequences. The program Matlnspector V2.250 was used USA), 5 ␮l of PCR product was denatured for 5 min at to identify potential protein-DNA binding sites.42 95°C with 20 ␮l of denaturing mix (90% formamide, 0.5 × TBE, 0.02 N NaOH, 0.1% xylene cyanol, 0.1% Statistical analysis bromocresol purple) and then quenched on ice. The The association to the SYNJ1 SNPs was tested using sample was loaded onto 4–20% non-denaturing acryla- the Pearson ␹2 test (2 × 2 table for allele frequency) and mide minigels (1 mm thick) containing 4% glycerol in a ␹2 test for independence (3 × 2 table for genotypes). 1.25 × TBE buffer. A control sample containing non- A statistical program, STATA, was used to compute denatured DNA was used to identify bands that corre- the ␹2 values. All P-values are 2-tailed. Haplotype sponded to annealed double stranded DNA. The analysis was conducted using the EH program from the samples were subjected to electrophoresis at 200 V at LINKAGE package. 4°C until the xylene cyanol dye reached the bottom of the gel. The temperature of the gel was maintained Results using a circulating, temperature controlled water bath. Following electrophoresis, the gel was stained with The 21q22-linked SYNJ1 gene spans more than 70 SYBR II dye according to the manufacturer’s instruc- kilobases (kb) and contains more than 30 exons. The tions (Novex) and photographed under UV light. The brain-specific 145-kD isoform and the 170-kD isoform minigel system was generally used for PCR products are generated by an alternative splice at exon 32. The smaller than 225 nucleotides. For larger samples, brain-specific exon 32 contains an inframe stop codon between 225–300 nucleotides, SSCP screening was that results in a relatively short proline-rich C-terminal performed using a standard long sequencing apparatus end. To generate the 170-kD isoform, there is an alter- with radio-labelled PCR products. PCR products were native splice in which the brain-specific exon is labelled by reducing the dCTP final concentration from bypassed and a large 3Ј exon is incorporated. This 200 ␮M to 100 ␮M and adding 0.25 ␮l 32P-dCTP results in a protein with a different proline-rich C-ter- (3000 Ci mM−1). For most PCR products larger than 300 minal end containing 266 additional amino acids. The nucleotides, radio-labelled PCR products were digested proline-rich terminus contains several SH3 domain- with a convenient restriction to generate binding sites which are involved in target specificity.34 smaller fragments. The exception was the exon 28/29 The Sac1 and inositol 5-phosphatase domains are simi- primer set which generates a 370-bp PCR product that lar in both the brain-specific and ubiquitous forms. contains two common polymorphisms and two rare However, alternative splicing at other exons may create mutations that could all be resolved without restriction additional diversity in the catalytic domains. The digestion. The samples were denatured and subjected translation initiation site is found in exon 2. In this to electrophoresis at 4°C through long 5% acrylamide study, the 32 exons that encode the brain-specific iso- gels containing 5% glycerol until the xylene cyanol form (GenBank accession number AF009039), and tracking dye migrated a distance of 30–40 cm. Gels 839 bp of 5Ј-flanking DNA, were screened for were dried and exposed to X-ray film at −70°C with mutations using SSCP analysis (see Table 1 for list of intensifying screens for approximately 18 h. primers). The DNA samples used in the initial screen- DNA samples with SSCP band shifts were subjected ing were derived from 45 unrelated patients with BPI to DNA sequence analysis. This was performed on pur- disorder. ified PCR templates using fluorescent-labelled dideoxy A total of 11 mutations were detected in the SSCP sequencing and an Applied Biosystems model 377 screening. Of these, seven were quite rare and will be sequencer. prior to sequencing, the PCR products were described first (Table 2). One rare variant was found in purified using a commercially available column to sep- the 5Ј-flanking region, 1898 nucleotides upstream from arate unused primers and nucleotides from the PCR the translation start site (−1898TϾC). The mutation reaction (Qiagen, Valencia, CA, USA). The sequencing was found in only one out of 140 bipolar subjects and data were compared with the available published zero out of 148 controls. Although the functional role sequence information using BLAST. Both DNA strands of this mutation is not known at this time, it occurs in were sequenced. a putative E-box consensus, a for many All genotypes were performed by SSCP and scored basic helix-loop-helix (bHLH) proteins (Figure 1b).43 independently by two investigators (TS and HL). Rep- Using the exon 1 primer set, a rare CϾA transversion resentative radiolabeled SSCP gels used for genotyping was found in a GC-rich intron sequence 58 nucleotides

Molecular Psychiatry Mutation analysis of SYNJ1 T Saito et al 390

Figure 1 (a) SSCP. Representative autoradiograms of SSCP gels used to genotype the common polymorphisms in exon 8, IVS12, IVS28 and IVS29. Band patterns for homozygotes and heterozygotes shown. Both the IVS28 and IVS29 polymorphisms were identified on the same SSCP gel. The genotypes for these are shown at the top and bottom of the SSCP gel, while the haplotypes generated by combining the IVS28 and IVS29 alleles are shown at right. The haplotype generated by IVS28G and IVS29T was not detected. All genotypes were confirmed by sequencing. Autoradiograms shown for each polymorphism taken from same gel, although lanes shown were not necessarily contiguous. (b) Sequence of selected SYNJ1 mutations. Putative promoter mutation (−1898TϾC) at E-box binding site consensus (CANNTG) in bold type. IVS1+58CϾA mutation in GC-rich region (shown in bold) alters one of the two putative Sp1 consensus sequences (underlined). IVS6−49GϾA mutation showing putative native breakpoint region underlined; the mutation leads to a 6/7 match (bold type) for branchpoint consensus YNYYRAY (Y = pyrimidine, R = purine, N = any nucleotide, A = adenine). PPT is double underlined. Bottom of figure shows IVS12+15delT,+17delT. The poly AT-tract is underlined and the deleted thymidine residues at positions 15 and 17 relative to splice junction are shown in bold type.

from the intron 1 (intervening sequence 1 or IVS1) mutation. The siblings, obtained from the The National splice donor site (IVS1 + 58CϾA). This mutation was Institutes of Mental Health Bipolar Genetics Initiative found in only one patient with BPD out of 143 unre- DNA repository, are the offspring of parents who both lated subjects analyzed and no controls out of 148 ana- have psychiatric illnesses. lyzed. There is an 18/20 and a 52/60 GC stretch in the Two rare mutations were found with the exon 7 wild-type sequence and 17/20 and 51/60 in the mutant primer set. One is an AϾG transition in intron 7, 43 (Figure 1b). The mutation occurs in a consensus bind- nucleotides from the exon 7 splice donor site ing site for the transcription factor Sp1. This patient (IVS7+43AϾG). This SNP was found in six out of 149 had one sibling with BPD who also inherited the unrelated bipolar patients and five out of 148 controls.

Molecular Psychiatry Mutation analysis of SYNJ1 T Saito et al 391 Table 2 SYNJ1 mutations

Primer Variant Comments Freq. cont Freq. BPD pro 5 −1898TϾC possible E-box consensus in 0/148 1/140 promoter exon 1 IVS1+58CϾA 58 bp from splice donor, SP1 0/148 1/143 consensus exon 7 IVS6−49GϾA 6/7 breakpoint consensus 0/148 2/149 match exon 7 IVS7+43A−ϾG 43 bp from splice donor 5/148 6/149 exon 8 F286L conservative amino acid 0/148 1/147 substitution exon 8 K295R conservative amino acid Table 3 Table 3 substitution exon 12 IVS12+15delT, +17delT AT-rich region at splice donor Table 3 Table 3 exon 28/29 IVS27−29TϾG 29 bp from splice acceptor 2/139 0/145 exon 28/29 IVS27−33CϾA 33 bp from splice acceptor 1/139 0/145 exon 28/29 IVS28−7GϾT polypyrimidine tract Table 3 Table 3 exon 28/29 IVS29+14CϾT 14 bp from splice donor Table 3 Table 3

Summary of mutations. Frequencies only shown for rare alleles. See Table 3 for analysis of common variants.

One of the six bipolar patients had a sibling with BPD tive branchpoint consensus and the PPT in the 3Ј end available for analysis who was found to also have of intron 27 and are not expected to affect these regu- inherited the mutation. Another proband has two sib- latory domains. Only one control out of 139 analyzed lings with BPD only one of whom inherited this and one bipolar patient out of 145 analyzed inherited mutation. The intron 7 SNP does not appear to alter the −33 mutation while two controls and no patients the sequence of any known functional domain. with BPD inherited the −29 mutation. The other mutation detected with the exon 7 primer The compilation of all the rare alleles is shown in set was found in intron 6, 49 bp from splice acceptor Table 2. Eight rare alleles were found in 148 control site (IVS6−49GϾA). This was found in two patients subjects compared with 12 out of 149 bipolar subjects. with BPD and no controls. The mutation results in a However, when the rare mutations are sub-divided into partial match (6/7) to a consensus branchpoint those more likely to have functional significance, based sequence 19 nucleotides upstream from the putative on whether or not the mutation occurs in a known regu- native branchpoint sequence in this intron (Figure 1b). latory region, including −1898TϾC, IVS1+58CϾA, The branchpoint consensus (YNYYRAY, where IVS6−49GϾA and F286L, there were five rare alleles Y = pyrimidine, R = purine, N = any nucleotide, found in patients with BPD and no controls (Table 2). A = adenine) is important in intron excision at the 3Ј However, the number is too small for a valid statisti- splice acceptor site.44 The putative natural branchpoint cal comparison. site is also a 6/7 match to the consensus. Whether the Four of the mutations we detected were very com- −49 mutation results in the creation of a ‘cryptic’ mon thereby allowing for a more valid comparison branchpoint site and influences splicing remains to between bipolar and control subjects using a case con- be determined. trol analysis. One is the exon 8 K295R variant for Two SYNJ1 coding variants were found in exon 8, which an allele frequency of 0.32 was found in both located in the Sac1 homology, at codon 286 controls and bipolar patients (Table 3, comparison (TTTϾCTT, pheϾleu, F286L) and codon 295 between controls and BPD, alleles: ␹2 = 0.001, P = 0.98, (AAA−ϾAGA, lysϾarg, K295R). Although both are 1 df; genotypes ␹2 = 0.23, P = 0.89, 2 df). conservative amino acid changes, pheϾleu and The two common SNPs detected using the exon lysϾarg substitutions have been found that alter pro- 28/29 primer set included a GϾT transversion in a PPT tein function.45–47 The F286L variant was only found at the intron 28 splice acceptor site, seven nucleotides in one out of 147 bipolar subjects and zero out of 148 from exon 29 (IVS28−7GϾT), and a CϾT transition in control subjects. This individual had two siblings with intron 29, 14 nucleotides from the exon 29 splice donor BPD but neither one inherited this mutation. The (IVS29+14CϾT). Since PPTs bind to a number of pro- analysis of K295R, a very common variant found at a teins, including heterogeneous ribonucleoprotein frequency of 0.32 is discussed later in this section. (hnRNP) and polypyrimidine tract binding protein Two common and two rare mutations were found (PTB), and are important in removing the intron from when the exon 28/29 primer set was analyzed. The two pre-mRNA and in alternative splicing, we viewed the rare mutations are a CϾA transversion and a TϾG IVS28, −7GϾT SNP, which increases the number of transversion 33 bp and 29 bp, respectively, 5Ј to the pyrimidines in the PPT, as a possibly functional exon 28 splice acceptor site (IVS27−33CϾA, and mutation.48–50 However, similar to the K295R variant, IVS27−29TϾG). These were found between the puta- the frequency of both the IVS28, −7GϾT and IVS29,

Molecular Psychiatry Mutation analysis of SYNJ1 T Saito et al 392 Table 3 Case control comparison of common SYNJ1 poly- quencies between bipolar subjects and controls for all morphisms of the other common polymorphisms, there was a slight increase in allele 2 in bipolar subjects that fell Controls Bipolar short of statistical significance (Table 3, alleles: ␹2 = 2.45, P = 0.12, 1 df, odds ratio = 1.3 95% CI = 0.93– codon 295 (K295R) 1.79; genotypes ␹2 = 2.29, P = 0.33, 2 df). genotypes There was a 27% increase in the number of bipolar AA 68 (0.46) 66 (0.45) subjects homozygous for allele 2, compared with con- AG 64 (0.43) 67 (0.46) trols, without any difference in the percentage of het- GG 16 (0.11) 14 (0.10) erozygotes. Consequently, if this allele proves to play alleles any role in the development of BPD in a subset of A 200 (0.68) 199 (0.68) G 96 (0.32) 95 (0.32) patients, its pattern of inheritance will be as an autoso- IVS28−7GϾT mal recessive trait. genotypes Haplotypes were generated using the K295R, GG 66 (0.47) 66 (0.46) IVS12+15delT,+17delT, and IVS28−7TϾG polymor- GT 59 (0.43) 66 (0.46) phisms. There was no significant departure from TT 14 (0.10) 13 (0.09) Hardy–Weinberg equilibrium for any of the 36 poten- alleles tial genotypes generated from the combined alleles. A G 191 (0.69) 198 (0.68) borderline significant result was found with a haplo- T 87 (0.31) 92 (0.32) type generated from IVS12 allele 2, and exon 8 allele IVS29+14CϾT G (dominant disease model: P = 0.075 for 80% pen- genotypes CC 87 (0.625) 91 (0.627) etrance and 0.072 for 10% penetrance). CT 44 (0.316) 49 (0.337) TT 8 (0.057) 5 (0.034) Discussion alleles C 218 (0.78) 231 (0.80) Based on theoretical considerations and some experi- T 60 (0.22) 59 (0.20) mental findings, we have recently begun to analyze IVS12+15delT, +17delT) genes involved in synaptic vesicle function as candi- genotypes dates for BPD and SZ. In a previous study, an SNP in 11 50 (0.34) 40 (0.27) the intron 7 PPT of the SYBL1 gene, a synaptobrevin 12 66 (0.45) 67 (0.45) analog that maps to Xq28, was found at a slightly 22 32 (0.22) 41 (0.28) alleles higher frequency in males with BPD compared with 18 1 166 (0.56) 147 (0.496) controls. In another study (in press), we identified a 2 130 (0.44) 149 (0.503) mutation in the promoter of the SNAP29 gene that was associated with SZ. SNAP29, a member of the SNAP25 Genotypes and alleles for the four common polymorphisms family of genes, maps to 22q11 in a region deleted in detected in the SYNJ1 gene. Number of subjects with each approximately 70% of patients with velo cardio facial genotype and number of alleles are shown with frequency in syndrome (VCFS), a congenital disorder associated parenthesis. There were no significant differences in the allele with a high prevalence of psychiatric disorders includ- and genotype distributions except for a trend towards associ- ing BPD and SZ.13,16,51 Considering the high false posi- ation in the allele distribution for intron 12 mutation tive rate using the case control design, the findings ␹2 = = = = ( 2.45, P 0.12, 1 df, odds ratio 95% CI 0.93–1.79). with SYBL1 and SNAP29 should be viewed as very preliminary. In a recent analysis of the chromosome +14CϾT SNPs and the distribution of alleles and geno- 22-linked synapsin III gene in schizophrenics, no dis- types were very similar in controls and bipolar subjects ease-causing mutations were found.52 (Table 3, intron 28 alleles; ␹2 = 0.07, P = 0.80, 1 df; In the study reported here, we have extended our genotypes ␹2 = 0.92, P 0.86, 2 df; intron 29 alleles: investigation of genes involved in synaptic vesicle ␹2 = 0.13, P = 0.70, 1 df; genotypes ␹2 = 0.6, P = 0.63, function by analyzing SYNJ1, which encodes an inosi- 2 df). tol 5-phosphatase that plays a critical role in the endo- The only SYNJ1 mutation that showed a nominal dif- cytic phase of the synaptic vesicle recycling pathway. ference between bipolar subjects and controls was the Studies in mouse knockouts show that SYNJ1 is one detected using the exon 12 primer set. This is an needed for normal neuronal function. Homozygous interesting polymorphism in a poly AT tract that deletion in mice results in the accumulation of

begins 11 nucleotides downstream from the exon 12- (PI(4,5)P2) and a defect in endocytosis with clathrin- intron 12 border. The poly AT tract contains either coated vesicles accumulating in the inactive zone.31 25/26 (allele 1) or 23/24 (allele 2) adenine or thymidine Although basal glutamatergic transmission is not nucleotides. Allele 2 is generated by a deletion of the impaired, following a train of stimuli intended to make fifth and seventh thymidine residues in the poly AT the regeneration of active vesicles rate limiting, there tract, 15 and 17 nucleotides downstream from the 5Ј is a prolonged impairment of transmission caused by splice donor site (IVS12+15delT, +17delT) (Figure 1b). a decrease in the number of vesicles capable of In contrast to the marked similarity in the allele fre- returning to the active zone for exocytosis. Homo-

Molecular Psychiatry Mutation analysis of SYNJ1 T Saito et al 393 zygous deletion of Synj1 is lethal and no studies have mutation −1898TϾC, which occurs in a putative E-box been reported in heterozygotes. domain. Overall, five bipolar patients and no controls SYNJ1 maps to chromosome 21q22.2 and evidence were found to have one of these mutations. The possi- for linkage to this region in a subset of families with bility that F286L is relevant to BPD is diminished by BPD has been provided in several studies.22–28,53 Straub its absence in the proband’s two bipolar siblings. How- et al24 initially identified markers on 21q22 linked to ever, any potential functional allele should not be BPD, using the affected pedigree member method of immediately ruled out as a candidate in view of the analysis, with the most significant result obtained for high frequency of assortative mating and oligogenic a marker in the PFKL locus on 21q22.3, which is inheritance associated with BPD. approximately 11.6 million nucleotides telomeric to The only common polymorphism that showed some 23,24 SYNJ1 (Figure 2). A positive lod score in the same difference in allele frequency between controls and 53 region was also found by Gurling et al, and Smyth et bipolar patients was the intron 12 variant in which 27 al. In the NIMH bipolar initiative consortium, Detera- there was a 27% increase in homozygotes for allele 2 26 Wadleigh et al identified a number of markers over a in bipolar subjects compared with controls. However, broad range on 21q22, centromeric to PFKL where population stratification could account for this small excess allele sharing was detected. The markers that difference. had the most significant P-values in their affected sib The intron 12 variant is caused by the deletion of pair (ASP) analysis were D21S1254, D21S65, two non-adjacent thymidine residues in an AT-rich D21S1252 and D21S1440, which are all within 5 region near the exon 12 splice donor site. The func- million nucleotides telomeric to SYNJ1. The most sig- tional significance of the AT tract and the effect of the nificant result in their ASP analysis using an affected deletion polymorphism is not known at this time. model that included recurrent unipolar depression, as well as BPD, was obtained for a set of markers that also However, considering that ‘AT’ and ‘AU’ rich regions included D22S1270, which is 2.4 million nucleotides are known to bind homeotic proteins, act as enhancers, centromeric to SYNJ1. Since non-parametric methods and affect mRNA stability, functional significance is a 56–59 of linkage can only provide a rough estimate to the map distinct possibility. Whether or not the poly position of a trait, the relative proximity of SYNJ1 to AT(AU) tract in intron 12 and the different allelic vari- the linked markers suggests that it can be considered a ants we identified have an effect on SYNJ1 gene feasible candidate gene for 21q22-linked BPD. Several expression remains to be determined. negative results have also been reported for this region, A limitation of our study is that the SSCP method suggesting the possibility of genetic heterogeneity.54,55 used in our mutation screening may fail to detect 10– In our screening of the SYNJ1 gene, several alleles 20% of polymorphisms. Also, we have not yet ana- that may be functionally significant, including two lyzed the large terminal exon that encodes the 170-kd coding variants and several intron and promoter isoform, which is expressed ubiquitously. In addition, mutations occurring in putative regulatory regions, there may be additional regulatory regions, other than were identified. Of these, only the common exon 8 the 5Ј-promoter region, that may harbor functionally variant (K295R) and IVS28−7GϾT, which occurs in a significant mutations. Intron 1, for example, is very GC- PPT, were found in a sufficient number of subjects for rich and may contain DNA elements that regulate gene a valid comparison using the case control method. The expression. Considering the identification of a rare allele frequencies for these polymorphisms, as well as mutation that may affect the binding of a transcription another SNP, IVS29+14CϾT, which does not appear to factor, found in a single bipolar subject and an affected have functional significance, were very similar in both sibling, intron 1 deserves a more thorough analysis. bipolar and control groups. Although the results of this study are marginal, with Among the rare SNPs, several appear to be of interest the findings of a modest increase in the intron 12 based on the possibility that they may have functional deletion variant, and an increase in rare alleles that significance. These include IVS1+58CϾA, which may have functional significance, the SYNJ1 gene war- changes an apparent Sp1 consensus sequence, rants further investigation as a candidate for 21q22- − Ͼ IVS6 49G A, which may create a cryptic breakpoint linked BPD. consensus, the coding variant F286L, and the promoter

Figure 2 Map of chromosome 21q22 showing SYNJ1 and selected markers. Distance from centromere in megabases shown at bottom.

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Molecular Psychiatry