¹Department of Psychiatry, Johns Hopkins University School of Medicine, Baltimore, MD, USA

²Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD, USA

³Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, MD, USA

⁴National Institute of Diabetes and Digestive and Kidney Disease, National Institutes of Health, Bethesda, MD, USA

Correspondence to: C A Ross, or MG McInnis, Department of Psychiatry, Johns Hopkins University School of Medicine, 600 N Wolfe Street, Baltimore, MD 21287-7463, USA. E-mail: mmcinnis@jhmi.edu; caross@jhu.edu

Accession numbers for the sequences submitted to GenBank are AF149426-AF149699

we previously reported linkage between bipolar disorder and a region on human chromosome (hc) 18q21. to identify genes in this region, exon trapping was performed on cosmids isolated from an hc18-specific cosmid library (ll18nc02) using 47 sequence tagged site (sts) markers from 18q21 as hybridization probes. a total of 285 unique sequences (exons) were obtained from 850 sequenced clones. homology searching of the databases using ncbi's blast algorithms revealed that 31 exons have identity to known genes and/or ests, seven are identical to regions of finished genomic sequences in the 18q21 region, 20 have significant similarity (>30% sequence identity) to genes from human and/or other species, 19 were repetitive sequences, and 208 sequences (72%) are novel. Seventy per cent of the trapped sequences were predicted to be derived from genes using library screening and RT-PCR analyses. This represents an initial stage in characterizing genes in a susceptibility region for further study in bipolar disorder or other diseases that map to this region. Molecular Psychiatry (2000) 5, 502-509.

exons; exon trapping; chromosome 18; bipolar disorder

Introduction

Bipolar (BP) disorder is a severe psychiatric illness that is characterized by episodes of mania and depression. The etiology of the illness is presently unknown; however twin, adoption and family studies indicate a significant genetic component.¹ Linkage studies in bipolar disorder have identified several susceptibility loci on HC18.^{2,3,4,5,6,7,8,9} We reported evidence for a susceptibility locus on 18q21² which was confirmed in a second independent sample.³ The strongest evidence is in a 10-cM region defined by D18S41 and D18S60.

Bipolar disorder is a disease of complex inheritance and is likely to show considerable genetic heterogeneity. It is unlikely that linkage analysis will narrow the region definitively without substantial increases in the numbers of pedigrees in the analysis. Association studies using polymorphisms within expressed sequences will increase the power of the available samples.¹⁰ Further analysis of the regions of interest may include physical and transcription mapping, identification of the genes and corresponding full length cDNAs, and association analysis on selected genes.

As an initial step towards systematic analysis of the genes in this 18q21 region, we carried out exon trapping¹¹ using selected cosmid clones as input DNA. This method of gene identification is independent of the expression pattern. Here, we report the isolation of 285 exons on HC18, the mapping of 123 exons, and expression analysis of a subset of the trapped exons.

Materials and methods

Cosmid identification

The HC18-specific cosmid library (LL18NC02) and the corresponding high density filter (AD) containing all the clones were purchased from the Human Genome Mapping Project (HGMP) resource center in UK. This library was constructed at the Lawrence Livermore National Laboratory (http://www-bio.llnl.gov/genome/ html/cosmid.html). It contains 14 304 clones gridded in microplates, providing 5.7 ´ depth coverage of the chromosome. The average insert size of the clones is 40 kb. About 6% of the clones have no inserts, and 10% are from Chinese hamster genomic DNA.

Forty-seven sequence tagged sites (STSs) previously localized in the interval between D18S41 and D18S60 were selected to identify cosmid clones. PCR products of 27 markers comprising set 1 (Table 1) with unique sequences amplified from human genomic DNA were labeled with ³²P using a Rediprime labeling kit (Amersham cat. No. RPN1633). The 20 STSs of set II (Table 1) contained simple sequence repeats. Oligonucleotide primers representing the unique sequence of the STS were end-labeled with ³²P using T4 kinase to make the hybridization probes for set II. Five STSs were pooled to hybridize the AD filter successively. The filter was prehybridized in a RapidHyb buffer (Amersham, NJ, USA) at 65°C for 30 min (45°C for oligonucleotide probes), then ³²P-labeled probes were added to the prehybridization buffer and incubated for 2-3 h. After hybridization, the filter was washed in 2´ SSC/0.1% SDS for 15 min twice at room temperature, and washed in 0.1 ´ SSC/0.1% SDS for 30 min twice at 65°C (with 2 ´ SSC/0.1% SDS at 45°C for oligonucleotide probes). Positive clones were obtained by scoring the image of the overnight exposed X-ray film.

Exon trapping and sequence analysis

Cloning of cosmid DNA fragments into splicing vector pSPL3 was done as described by Chen et al.¹² Briefly, cosmids were grown individually in TB medium overnight, and 5-10 clones were pooled to isolate DNA. Each pool was digested with EcoRI and/or PstI restriction enzymes, and the fragments were cloned into pSPL3 in the appropriate cloning sites to make sub-libraries. Plasmid DNA isolated from each sublibrary was used to transfect COS7 cells that were passed 12 h before transfection and grown into 60-80% density. Total RNA was isolated from COS7 cells and the trapped sequences were amplified using an exon trapping system (Life Technologies cat. No. 18449-017). The RT-PCR amplified sequences were then cloned into vector pAMP10. Colonies were picked and arrayed in 384-well microplates. Vector pSPL3 derived sequences were eliminated using oligonucleotide hybridization as described by Chen et al.¹² Another oligonucleotide, COS1 (5' TTAAGTTATGACGAAGAA GAAC), was included to identify clones derived from vector lawrist16. Exon-containing clones were screened by PCR amplification of inserts using oligonucleotide primers SD2 (5' GTGAACTGCACTGTGAC AAGCTGC) and SA4 (5' CACCTGAGGAGTGAATT GGTCG). Sequences were determined on AB1377 automated sequencers at the Johns Hopkins Genetics Core Facility laboratories using SD2 as sequencing primer.

The derived sequences were entered into a Discovery Manager database¹³ and screened for redundancy using BLAST. Unique sequences were listed and scanned for sequence homologies in public databases at the National Center for Biotechnology Information (NCBI) using BLAST.¹⁴

Mapping the exons to 18q21

Oligonucleotide primers were designed for 194 exons and assayed on the GeneBridge4 (GB4) radiation hybrid panel¹⁵ and a contig of eight YACs localized in the 18q21 region. The GB4 panel was purchased from the HGMP biological resource center and Research Genetics, respectively. PCR was done in a 10-l reaction containing 25 ng GB4 hybrid DNA, 1´ PCR buffer (50 mM KCl, 10 mM Tris-base and 1.5 mM MgCl₂, pH 8.4), 1 l dNTPs of 10 mM, and 0.5 l each primers of 5 M, and 0.7 U Taq polymerase. The PCR cycles were set as follows: initial denaturation at 94°C for 5 min, followed by 35 cycles of 94°C for 20 s, 55°C for 20 s, 72°C for 30 s, and a final extension at 72°C for 7 min. Vectors generated by scoring the presence or absence of amplification in each hybrid were submitted to the web servers at HGMP (http://menu.hgmp.mrc.ac.uk/ menu-bin/RHyME/RHyME.pl) and the Sanger Center (http://www.sanger.ac.uk/RHserver/RHserver.shtml) to localize the exons to the chromosome.

Expression analysis

Twelve exons from the 18q21 region were used to screen the cDNA library. At least 1 ´ 10⁶ cDNA clones of a human fetal brain library (Clontech cat. No. HL3003a) were screened. The exons were labeled with ³²P using the Rediprime kit (Amersham). The filters were prehybridized in RapidHyb buffer (Amersham) at 65°C for 1 h, and the probes were added and incubated at 65°C overnight. After hybridization, the filters were washed in 2 ´ SSC/0.1% SDS at room temperature for 20 min, and repeated twice, then in 0.1 ´ SSC/0.1% SDS at 62°C for 30 min twice. The filters were exposed to X-ray films at -80°C overnight.

PCR reactions were also performed on phage DNA amplified from at least 1 ´ 10⁶ pfu clones from the fetal brain cDNA library. PCR was carried out in a 20-l reaction containing 50 ng of purified phage DNA as templates, 1 ´ PCR buffer (50 mM KCl, 10 mM Tris-base and 1.5 mM MgCl₂, pH 8.4), 1 l dNTPs of 10 mM, and 1 l each primers of 5 M and 0.7 U Taq polymerase.

In addition, 34 exons mapped in the 18q21 region were tested for expression by RT-PCR using cDNA template derived by reverse-transcription of fetal brain polyA+ RNA (Clontech cat. No. 6525-1). Reverse transcription was done using the Marathon cDNA amplification kit (Clontech cat. No. K1802-1) and its protocol PT1115-1, and the same RNA was used as control in the absence of reverse transcriptase.

Results

Identification of 285 exons

Cosmid clones from HC 18q21 were isolated from the LL18NC02 cosmid library by hybridization with 47 STS markers (see Methods and Table 1). These STSs were previously localized in the region between D18S41 and D18S60. A total of 350 positive cosmids were isolated. Cosmid DNA from pools of 5-10 clones was subcloned into vector pSPL3 for exon trapping.¹¹

Oligonucleotide primers were designed to eliminate clones containing cryptic spliced sequences from the vector pSPL3 by a hybridization screen.¹² Eight hundred and fifty pAMP10 clones containing potential exons were sequenced on one strand using the sequencing primer, SD2. The sequences were entered into a Discovery Manager (DM) database.¹³ There were 285 unique sequences from the total of 850 sequenced clones; BLAST analysis of the DM database identified an average of 3´ redundancy for each of the 285 unique sequences.

The size of the unique trapped sequences ranged from 25-406 bp, mean = 112 bp. The GC content of the trapped sequences (51%) was similar to that of cDNA rather than genomic DNA. Eighty-one per cent of the sequences (230 out of 285) were consistent with open reading frames (ORF) throughout, ie no evidence of stop codons in any of the reading frames. The size range, GC content and ORF frequency of the trapped exons are similar to previously published data from exon trapping.^11,12 Homology searches for all sequences were performed against the public databases (all non-redundant GenBank CDS translations+PDB+Swiss Prot+PIR+PRF) with NCBI's BLAST algorithms.¹⁴ Table 2 lists the significant findings of BLAST search results. Fifteen sequences were identical to seven previously cloned genes and corresponding ESTs in the region (Table 2A), including FECH, SLAT8, P15, SMAD7, KIAA0606, KIAA0439, and ASNRS. Sixteen exons were identical to ESTs (Table 2B). Seven exons identified portions of finished genomic sequences in the 18q21 region (Table 2C). Nine exons show similarity but not identity to genes from human or other species (Table 2D). We used the probability of 10^-3 as the cutoff point (or >30% identity in predicted amino acid sequences) for significance in homology searches. Nineteen sequences recognized Alu or other repeats. The remaining 219 sequences are novel. Two hundred and seventy-four sequences 50 bp were submitted to GenBank (accession Nos: AF149426-AF149699).

Mapping exons to the region of 18q21

The GeneBridge 4 (GB4) mapping panel¹⁵ and a contig of eight YACs localized between D18S41 and D18S60 were used to map exons with PCR amplification (see Methods). PCR primers were designed for 194 exons longer than 70 bp. There were 71 primer sets that did not amplify human DNA (nine amplified hamster DNA), leaving 123 primer pairs for mapping. PCR analysis localized 25 exons to the 18q21 YAC contig. The remaining 98 exons were mapped to chromosome 18 using the GB4 panel, of which 35 localized to 18q21. None of the exons mapped elsewhere in the genome. Thus a total of 60 of the exons were mapped to the region of 18q21 between markers D18S41 and D18S60 (Figure 1).

Expression analysis

In addition to database searching, three experimental approaches were used to estimate how many putative exons derived from expressed genes. The number of exons chosen for expression analysis in the following three different approaches was arbitrary. A total of 94 exons were analyzed; all 60 exons whose location was confirmed to 18q21 were selected for expression analyses, and an additional 34 were selected at random from the remainder of the trapped exons. First, a fetal brain cDNA library was screened by hybridization using two pools of exons. Pool 1 contained exons e1a6, e1b1, e1b5, e2e11, e2g2 and 1d23, and pool 2 included exons 10a3, 10b16, 10d9, 10d14, 10h2 and 6f8. Pools 1 and 2 identified 52 and 65 strong positives, respectively. Positive phage plaques identified by hybridization were then screened by PCR. All 12 of the exons tested identified two or more cDNA clones. Second, 34 of the 60 exons from 18q21 were selected for RT-PCR analyses; 26 of 34 exons amplified a product from cDNA templates derived by reverse transcription of commercial fetal brain polyA+ mRNA (Clontech cat. No. 6525-1) (Table 3). All negative control samples showed no amplification when performing RT-PCR. Finally, the remaining 14 of the 60 18q21 exons were grouped with the additional 34 exons chosen at random from the remaining exons trapped from the whole of chromosome 18. Thirty-one of these 48 exons tested amplified phage DNA made from a fetal brain cDNA library (data not shown). In all, 94 different exons were tested in these paradigms (none was included twice), and 69 identified transcripts. Thus, we predict that at least 70% of the 285 unique exons trapping products were derived from genes.

Discussion

Exon trapping¹¹ was used to identify novel sequences from chromosome 18 that are likely to be expressed in an effort directed towards identification of susceptibility genes for bipolar disorder on 18q21.² Two hundred and eighty-five unique exons were trapped from 350 cosmids isolated using 47 STS mapped markers between D18S41 and D18S60. An output rate of 0.8 unique exon per cosmid was similar to other long range exon trapping.^12,16 The background of vector-derived false positive pAMP subclones varied between 20-45% in different trapping pools as seen by oligonucleotide hybridization (not shown), a similar background to that observed in previous experiments.¹² The frequency of repetitive sequences in the trapped exons was 6.7% (19 of 285) in this experiment, compared to the 8.9% reported by Chen et al.¹²

There were 194 exons >70 bp and these were used in the mapping experiments; 123 were successfully mapped to chromosome 18 and of these 60 were mapped to 18q21. The remaining 71 primer pairs did not amplify human DNA. This could be due to at least two reasons: (1) the trapped sequence contains two exons and genomic DNA is not amplified by PCR conditions designed for short fragments; (2) the trapped sequence comes from the rodent background of the cosmid library, nine of the 71 primer pairs amplified hamster DNA. All the remaining 123 exons tested mapped to HC18, and no exon mapped elsewhere in the genome. Based on this ratio (60/123) it is estimated that up to 142 (50%) of the 285 unique trapped exons are from 18q21.

It is difficult to determine exactly how many genes are represented by this collection of trapped exon sequences. However, based on the seven genes identified by 15 exons, it was extrapolated that on average two exons hit per gene; and therefore 285 exons may represent up to 140 genes. As suggested by the mapping experiments, the 142 exons estimated to be from 18q21 could represent up to 70 genes.

Exons trapped from the same cosmid are likely to be from the same gene. Of the 60 exons mapped to the 18q21 region, 53 amplified (PCR) different cosmids used in the trapping experiment, seven exons hit three cosmids of the LL18NC02 library (data not shown). These seven exons were: 2D20, 2E19 and 2F21 from cosmid AD4014, 6F1 and 6F8 from cosmid AD35a20, E4D6 and E4D9 from cosmid AD22f23. In addition, six exons identified portions of three finished BAC sequences in the 18q21 region (Table 2C), these could be from the same gene as well. However, there is at least one example of a BAC containing two genes. Exon E2G2 (Table 2C) identified the BAC sequence, AC011331. This BAC sequence contains a novel gene (KIAA0439) that is homologous to the NEDD4 family.¹⁷ The full length cDNA for KIAA0439 has been cloned (Chen et al, manuscript in preparation). E2G2 is located 2.6 kb downstream of the last exon of the gene, KIAA0439 and is on its complementary strand. Extrapolation of these data suggests that the 142 exons (60-70 novel genes) in the 18q21 region, combining with the NCBI Unigene database of published ESTs and genes (ca 45), represents over 100 genes from this region, which approaches the estimated average number of genes for a 5-6 Mb region. Expression analysis indicated that at least two thirds of the exons were expressed in fetal brain. This number may increase if RNA from various regions of brain and other organs and several stages of development are used. To date fewer than a dozen genes have been identified for which the full length cDNAs are cloned. Our approach represents one method of identifying regional genes.

In summary, exons representing the majority of genes from the 18q21 region have been identified. This represents an initial step in the ongoing genetic analysis of a region of interest in complex disorders such as bipolar disorder. It is premature to specify which of the exons should be pursued as 'candidate' genes for full length cDNA development and extensive mutation detection in bipolar disorder. The selection of such candidates will be contingent on expression studies and homologies with gene classes that bear physiological relevance to bipolar disorder. However, the present exon sequence information has a number of important uses: (1) it provides probes for screening cDNA libraries to isolate full length cDNAs; (2) it provides template for screening the public sequence databases, in particular the finished genomic sequences for homology and 'in silico' gene identification, providing biological support to sequence algorithm gene searches; (3) it contributes to the detailed transcription map of the chromosomal region; and (4) the sequences from the cloned exons and related genes serve as reference sequences to identify polymorphisms that could be subsequently typed in the disease population of interest. Polymorphisms within these genes will be useful for further association studies. It has been observed that association studies using single nucleotide polymorphisms (SNPs) that are within genes have significantly more power than those that are outside of genes.¹⁸ These novel sequences, representing an estimated 70 genes, from 18q21 will be useful in the study of bipolar disorder and other diseases that map to this region.

Acknowledgements

This study is supported by the Theodore and Vada Stanley Foundation; NIH grants to CAR (MH50763), MGM (MH01088) and JRD (MH42243); and National Alliance for Research on Schizophrenia and Depression (NARSAD) grant (JRD, MGM). We thank Drs Russell L Margolis and Virginia Willower for critical reading of the manuscript. We also thank Dr Russell L Margolis and John Kleiderlain for providing us with a set of filter lifts of a cerebellum cDNA library, and Drs OC Stine and A Heinzer for YAC contig construction.

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Figure 1 Mapping of 60 exons to the 18q21 region linked to bipolar disorder. GeneBridge4 (GB4) radiation hybrid mapping panel and eight YACs previously localized in the 18q21 region were used as PCR amplification materials. There are gaps of unknown size between the YACs. Placement of each exon in the map is according to results from GB4 mapping and PCR amplification of the YACs. Localization of exons E2G2, 6G6, 9G12, 9G14, 9G15, 9124 and 9K15 are also based on sequence identity to finished genomic sequences (AC011331, AC007628, AC006203 and AC006221) in GenBank, and the corresponding finished sequence contigs are shown as dark bars. Exons in parentheses are mapped both on GB4 and YACs.

Table 1 List of 47 STS markers used in cosmid isolation

Table 2 Trapped exons identical/homologous to genes, ESTs, finished genomic sequences in GenBank

Table 3 Expression analysis of 34 exons with RT-PCR