Gene 270 (2001) 69±76 www.elsevier.com/locate/gene RAI1 is a novel polyglutamine encoding gene that is deleted in Smith±Magenis syndrome patients q Peter Seranski a, CeÂline Hoff a, Uwe Radelof b, Steffen Hennig b, Richard Reinhardt b, Charles E. Schwartz c, Nina S. Heiss a, Annemarie Poustka a,* a Abt. Molekulare Genomanalyse, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany b Max Planck Institut fuÈr Molekulare Genetik, Ihnestrab e 73, 14195 Berlin, Germany c Greenwood Genetic Center, Greenwood, SC 29646, USA Received 3 November 2000; received in revised form 2 February 2001; accepted 27 February 2001 Received by M. D'Urso Abstract The human chromosomal band 17p11.2 is a genetically unstable interval. It has been shown to be deleted in patients suffering from Smith± Magenis syndrome. Previous efforts of physical and transcriptional mapping in 17p11.2 and subsequent genomic sequencing of the candidate interval allowed the identi®cation of new genes that might be responsible for the Smith±Magenis syndrome. In this report, one of these genes named RAI1, the human homologue of the mouse Rai1 gene, has been investigated for its contribution to the syndrome. Expression analysis on different human adult and fetal tissues has shown the existence of at least three splice variants. Moreover, the most interesting feature of the gene is the presence of a polymorphic CAG repeat coding for a polyglutamine stretch in the amino terminal domain of the protein. q 2001 Elsevier Science B.V. All rights reserved. Keywords: Smith±Magenis syndrome; Schizophrenia; Gene isolation; CAG-Repeat; Polymorphism; Human Chromosome Band 17p11.2 1. Introduction The Smith±Magenis syndrome (Smith et al., 1986) is genetically characterized by an interstitial deletion in the human chromosomal band 17p11.2. The deletion is associated with varying phenotypes including brachycephaly, prognathism, a hoarse voice, speech and growth delay as well as mental retardation, opthalmological abnormalities and REM-sleep disturbances. The broad variance of phenotypes was shown in several studies (Greenberg et al., 1991, Abbreviations: ALDH10, Fatty aldehyde dehydrogenase; ALDH3, Aldehyde dehydrogenase 3; DRPLA, Dentatorubral-pallidoluysian atrophy; FLI1, Drosophila Flightless-1 gene; LLGL1, Drosophila lethal-2-giant larvae gene; MFAP4, Micro®bril-associated protein 4; PAC, P1 arti®cial chromosome; RAI1, Human homologue of the mouse Rail (Retinoid acid induced 1)-Gene; REM, Rapid eye movement; SBMA, Spinal and bulbar muscular atrophy; SCA, Spinocerebellar ataxia types; SHMT1, Cytosolic hydroxymethyltransferase; SMS, Smith±Magenis syndrome; SREBF1, Sterol regulatory binding protein; TOP3, Human topoisomerase III; YAC, Yeast arti®cial chromosome q EMBL-Accession-No.: AJ271790, AJ271791. * Corresponding author. Tel.: 149-6221-424742; fax: 149-6221423454. E-mail address: a.poustka@dkfz.de (A. Poustka). 1996) and it is speculated, that the size of deletion correlates with the severity of the phenotype. Chen et al. (1997) have shown, that the deletion in the 17p11.2 band in SMS patients occurs between two ¯anking repeat gene clusters. There is further evidence that other complex sequence repeats are located in the 17p11.2 interval (Seranski et al., 1999). But it is still unclear, if the SMS phenotype is caused by the fusion of different genes from the repeating gene clusters or by the loss of one or multiple genes in the context of a contiguous gene syndrome. It is noteworthy, that also duplications in several patients with SMS phenotype were detected (Brown et al., 1996; Potocki et al., 2000). The same sequence repeat clusters are also suspected to be responsible for isochromosome i(17q) formation observed in medulloblastomas and different leukaemias (Scheurlen et al., 1996; Fioretos et al., 1999). The physical mapping of the unstable 17p11.2 interval deleted in SMS patients was based either on radiation hybrid maps (Wilgenbus et al., 1996), YAC contigs (Chen et al., 1997) or on PAC and cosmids (Wilgenbus et al., 1997; Seranski et al., 1999). This had led to the identi®cation of new genes and ESTs in the deletion interval. But still, the region remains incompletely mapped due to its complicated genomic architecture. 0378-1119/01/$ - see front matter q 2001 Elsevier Science B.V. All rights reserved. PII: S 0378-111 9(01)00415-2 70 P. Seranski et al. / Gene 270 (2001) 69±76 A number of genes have been discussed as potential candidate genes for Smith±Magenis syndrome. Genes known to map in the SMS deletion interval include the human homologue of the Drosophila lethal-2-giant larvae gene (LLGL1) (Strand et al., 1995; Koyama et al., 1996), the human homologue of the Drosophila Flightless-1 gene (FLI1) (Chen et al., 1995), the micro®bril-associated protein 4 (MFAP4) (Zhao et al., 1995), a sterol regulatory binding protein (SREBF1) (Hua et al., 1995), a cytosolic hydroxymethyltransferase, (SHMT1) (Elsea et al., 1995), human topoisomerase III (TOP3) (Elsea et al., 1998; Hanai et al., 1996), aldehyde dehydrogenase 3 (ALDH3) (Hiraoka et al., 1995), and a fatty aldehyde dehydrogenase (ALDH10) (De Laurenzi et al., 1996). In addition, a large number of ESTs with no homology to known genes or proteins were identi®ed between the two markers D17S805 and D17S953 which lie in the central SMS deletion interval (Chen et al., 1997; Elsea et al., 1997). The aim of this study was to characterize new genes using the results of previous gene isolation strategies and the genomic sequences of the candidate region. We therefore started to sequence the physically mapped PACs and cosmids, that were shown to be deleted in SMS patients. The use of gene prediction programs led to the identi®cation of a novel human gene on one PAC that showed signi®cant similarity to a well characterized mouse gene. Here we describe the identi®cation, veri®cation and further characterization of the novel human gene, RAI1. Its mouse homologue, Rai1, was originally cloned from the mouse P19 embryonic carcinoma cell line, a multipotent early embryo cell line (Imai et al., 1995). Furthermore, one shorter allele of the identi®ed CAG trinucleotide polymorphism of RAI1 has recently been shown to be associated with schizophrenia (Joober et al., 1999) and Hayes et al. (2000) suggest a possible involvement of the RAI1 CAG repeat in the pathogenesis of spinocerebellar ataxia type 2 (SCA2). 2. Material and methods 2.1. SMS patients Blood samples and lymphoblastoide cell lines of 17 patients with Smith±Magenis syndrome were collected by one of us (CES) as part of an ongoing study into the etiology of SMS. All patients have a deletion of 17p11.2 detected using standard high resolution karyotyping. The deletion was con®rmed by FISH analysis using a SMS speci®c cosmid probe. The cells were cultivated in suspension at 378C and 5.0% CO2 in RPMI1640-media, 20% FCS, 2 mM glutamin, 100 U/ml penicillin and 100 U/ml streptomycin. 2.2. Genomic sequencing The genomic sequence of the PAC clone I13281 was obtained applying the PrOF-method for sequencing with low redundancy as described (Radelof et al., 1998). Brie¯y, all shotgun clones were PCR ampli®ed, spotted onto nylon membranes and sequentially hybridized to a specially designed set of 100 short oligonucleotides. This creates oligonucleotide-®ngerprints, which are used to select optimal sets of shotgun clones prior to sequencing. The sequencing reactions are carried out using the dye primer technique on an ABI catalyst robot using 1 ml of the PCR product and 3 ml of the ThermoSequenase mix (Perkin±Elmer) for each of the four A;C;G;T reactions. Energy transfer primer (0.1 pmol for A, C and 0.2 pmol for G, T reactions, respectively) M13(240) or M13(228) were added to the ThermoSequenase mix before starting the sequencing run. Samples are pooled and precipitated according to ABI's instructions and analyzed on an ABI 377XL DNA sequencer. Data were processed using ABI's sequence analysis software versions 3.0 and 3.1, but with the Perkin±Elmer manual lane tracking kit according to the manufacturer's instructions. 2.3. RACE For RACE experiments, fetal brain Marathon cDNA (Clontech) was used. The primary RACE PCR reactions were carried out in a ®nal volume of 50 ml containing 5 ml of diluted marathon cDNA, 10 pmol AP1 primer (Clontech), 10 pmol gene speci®c primer, 50 mM Tris±HCl (pH 9.3), 16 mM (NH4)2SO4, 2 mM MgCl2, 200 mM each dNTP and 0.5 ml of 19:1 (Taq:Pfu) polymerase mix (Taq: 5 U/ml Perkin±Elmer; Pfu: 2 U/ml, Stratagene). After an initial denaturation for 3 min at 948C, ®ve cycles consisting of 948C for 15 s, 748C for 4 min were carried out, followed by another ®ve cycles consisting of 948C 15 s, 728C 4 min and ®nally another ten cycles consisting of 948C 15 s, 708C 4 min. Speci®c ampli®ed PCR products were re-ampli®ed with the same PCR conditions as above by using AP2 primer (Clontech) and gene speci®c primers. The marathon cDNA was replaced by 10 ng of primary RACE PCR product. Products were separated by agarose gel electrophoresis and transferred onto nylon membranes (HybondN 1, Amersham) by southern blotting. Speci®c ampli®cations were identi®ed by hybridization with terminally labeled oligonucleotides as probes. Speci®c PCR products were sequenced using the automated DNA sequencers and dye terminator chemistry (Amersham). The gene speci®c primers for the 5 0 -RACE were HSGT1B1 5 0 -ACA GGT GGA CAC GGA CTC GG-3 0 , HSGT1B2 5 0 -TCA CCC GCC ACG AAC TTG G-3 0 , HSGT1B4 5 0 -CCC CTT TGC TGA TGC CAT CTG AGG-3 0 , HSGT1B16 5 0 TTA CAG GGT GAA GAG GTG GTG TTC-3 0 . The gene speci®c primers for the 3 0 -RACE were HSGT1F11 5 0 -TGC TCC CTG AAT CCT GCA CAG-3 0 , HSGT1F15 5 0 -TGC AAG CGG CTG AGG TCA G-3 0 , HSGT1F16 5 0 -GCG GGT GGA GAA GCG AGA C-3 0 , HSGT1F12 5 0 -CCT ATA AGA GTT GCA CAG CAC CGA C-3 0 . 2.4. Expression analysis For the rapid expression analysis, EST-PCR primers from P. Seranski et al. / Gene 270 (2001) 69±76 71 Fig. 1. Genomic Structure of RAI1. The RAI1 predicted protein is encoded by eight exons, seven of them predicted by Genscan. The 5 0 UTR is not predicted and the 3 0 UTR with the polyadenylation signal is only 267 bp. The combination of the gene prediction results with cDNA sequences obtained from the cDNA selection, RT-PCR and RACE products led to the assembly of 5667 bp with an open reading frame of 5589 bp coding for 1863 amino acids. It contains one polyserine stretch with the structure [S]3A[S]8 in the carboxyterminal end and a polyglutamine stretch in the amino terminal end. The polyglutamine stretch is encoded by a polymorphic CAG repeat and lies within a glutamine rich domain with a total of 27 glutamine residues. Three RAI1 protein domains (aa 1268± 1298, aa 1553±1598, aa 1693±1725) show similarity (77, 45, 57%, respectively) to the human stromelysin PDGF responsive element binding protein transcription factor (GB U20282). The detailed exon-intron structure with the underlying genomic sequence is described under EMBL-Accession-No: AJ271791. cDNA sequences were generated (PS14E15F 5 0 - AACACC ACC TCT TCA CCC TGT AAG-3 0 , PS14E15B 3 0 -CCT TCT CCT TGA GTT TTG GCT TC-3 0 ) and tested on the adult human and fetal human multiple tissue cDNA panel (Clontech) using the Advantage cDNA PCR kit (Clontech). PCR-reactions (30 cycles) were carried out in a ®nal volume of 50 ml according to the supplier's instructions using a MJ Research Multicycler PTC200. Control PCR reactions were performed using the G3PDH control Amplimers according to the manufacturers instructions. In parallel, multiple-tissue northern blots (Clontech) were probed with a 1.6 kb RACE PCR-product in 6 £ SSPE, 5 £ SDS, 5 £ Denhardt's reagent for 20 h at 658C. The ®lters were washed at 658C 2 £ in 2 £ SSC, 0.1% SDS for 20 min. and 1 £ SSC, 0.1% SDS for 20 min and 0.5 £ SSC, 0.1% SDS. 2.5. CAG-repeat polymorphism DNA was isolated either from cell lines of SMS patients or from peripheral lymphocytes using standard methods (Sambrook et al., 1989). CAG repeats were ampli®ed by PCR using speci®c primers HSGT1CAGPF1, 5 0 -GGG GCA GCG GGT CCA GAA TC-3 0 and HSGT1CAGPB1, 5 0 -AGG GTT TCC TGG GCA TGG TGC-3 0 and an annealing temperature of 648C. PCR-reactions (30 cycles) were carried out in a ®nal volume of 50 ml containing 50 ng of template DNA, 10 mM Tris±HCl (pH8.8), 1.5 mM MgCl2, 50 mM KCl, 1% dimethylsulfoxide, 100 mM each dCTP, dGTP, dATP and dTTP and three units of Taq-Polymerase (Perkin± Elmer). PCR products were electrophoresed on denaturing 6% polyacrylamide gels and visualized by silver staining. To verify allele sizes and CAG repeat variants, all PCR products from SMS patients were directly sequenced on an ABI377 sequencer using big dye terminator chemistry. 3. Results and discussion 3.1. Genomic sequencing The PAC I13281 with a total genomic sequence length of 83 kb was assembled from six different contigs. The combined application of three different gene prediction algorithms, Genscan, GRAIL2 and MZEF, led to the identi®cation of a part of the novel human gene in one 23 kb sequence contig (EMBL Acc. No. AJ271791). Blast analysis of the genomic sequence identi®ed similarities to a mouse gene, that is retinoic acid induced and speci®cally expressed in the neurons of mouse brain during development. The novel gene is named RAI1 for the human homologue of the mouse retinoic acid induced gene Rai1 (former Gt1, GB D29801). This Genscan prediction was not consistent with the predictions obtained using GRAIL2 or MZEF. Both programs did not predict the complete RAI1 coding region. The Genscan prediction was con®rmed by RT-PCR analysis using exon speci®c primers. 3.2. cDNA cloning and protein analysis The combination of the gene prediction results with cDNA sequences obtained from the cDNA selection, RTPCR and RACE products led to the assembly of 5667 bp with an open reading frame of 5589 bp coding for 1863 amino acids (Fig. 1). The non-predicted 3 0 end of the gene 72 P. Seranski et al. / Gene 270 (2001) 69±76 Fig. 2. Clustal alignment of human and mouse RAI1. The BOXSHADE alignment illustrates the amino acid identity between the human and the mouse Rai1 translated gene product. The initiating methionine of the human cDNA sequence corresponds to the start methionine of the mouse protein. All identical amino acids are marked with an asterisk in the consensus line. Conserved amino acid exchanges are marked with a P. Seranski et al. / Gene 270 (2001) 69±76 was isolated through cDNA selection (PS14E15, EMBL AJ230819, Seranski et al., 1999). The predicted protein consists of 1863 amino acid. It contains one polyserine stretch with the structure [S]3A[S]8 in the carboxyterminal end from amino acid 1516±1527 and a polyglutamine stretch in the amino terminal end. The polyglutamine stretch is encoded by a polymorphic CAG repeat and lies within a glutamine rich domain reaching from amino acid 239 to amino acid 314 with a total of 27 glutamine residues. The predicted protein is proline (11.8%), serine (11.4%) and alanine rich (9.1%). It is hydrophilic without any hydrophobic, potential transmembrane spanning domains. Three RAI1 protein domains (amino acid 1268±1298, amino acid 1553±1598, amino acid 1693±1725) show similarity (77, 45, 57%, respectively) to the human stromelysin PDGF responsive element binding protein transcription factor (GB U20282). Fig. 2 illustrates high homology between the mouse Rai1 and human RAI1 protein, the overall identity between the human and mouse protein is 76%. The carboxyterminus of the human protein extends over the mouse protein by 160 aa residues, taking this into account the identity is 84.5%. 3.3. CAG-polymorphism Besides the high homology to the mouse orthologue, overall the predicted RAI1 peptide sequence shows no signi®cant similarities to any other known proteins. The most prominent feature in the deduced amino acid sequence is a polyglutamine stretch encoded by a polymorphic CAG repeat at the amino terminal end of the protein. To investigate the role of the amino terminal polyglutamine encoding CAG repeat in the mechanism responsible for the Smith Magenis Syndrome, potential expansion of the repeat was analyzed in Smith±Magenis patients. PCR products were obtained with primers ¯anking the CAG repeat. The analysis of 17 SMS patients and 77 normal heterozygous controls con®rmed the heterozygous deletion in all patients, but no signi®cant ampli®cations of the CAG repeat were detected. In total, ®ve different alleles were detected by polyacrylamide gel electrophoresis and subsequent silver staining (data not shown). Sequencing of the different alleles con®rmed the CAG repeats ranging in size from 36 to 42 bp. The CAG repeats of 13 SMS patients were sequenced, but neither expansions nor frame shifts or point mutations were detected (Table 1). Table 1 Allele frequency detected after sequencing of the CAG repeat of RAI 1 in 13 SMS patients Allele Sequence Number of SMS patients A B C D CAGCAA(CAG)12CAA (CAG)13CAA (CAG)12CAA (CAG)14CAA 3 7 2 1 73 Expanded CAG repeats have been found as the causal mutation involved in a number of neurodegenerative diseases such as HD (The Huntington's Disease Collaborative Research Group, 1993), SCA (Spinocerebellar Ataxia Types 1, 2, 3, 6, and 7; Ban® et al., 1994) SBMA (Spinal and Bulbar Muscular Atrophy; La Spada et al., 1992), and DRPLA (dentatorubral-pallidoluysian atrophy; Koide et al., 1994). In these cases, there is an increase in the lengths of the CAG repeats from generation to generation which is associated with a concomitant increase in severity of the disease, a phenomenon referred to as anticipation. One of the characteristic features of SMS are the neurological abnormalities and mental retardation. Even though the phenomenon of anticipation has not been reported in SMS and clinical evaluation of the parents show no subtle phenotype of SMS, it nonetheless seemed a possibility that expansions of CAG repeats in RAI1 could play a role in the aetiology of SMS. For these reasons we decided to focus our mutation analyses on the CAG repeat of RAI1. Seventeen patients who showed LOH of one RAI1 allele, contained no expanded CAG repeats of the remaining allele. In one family (8228, 8229, 8230) patient 8229 exhibited a different, smaller allele than both parents (Allele A). It is interesting that this is the same RAI1 CAG-allele which was found to be signi®cantly associated with schizophrenic patients representing a group of responders to neuroleptics, although the relevance of this is presently unclear (Joober et al., 1999). Recently, the association of the RAI1 CAG repeat length with age of onset variability in spinocerebellar ataxia type 2 (SCA2) was shown (Hayes et al., 2000). Compared to the human RAI1 cDNA which contains up to 13 CAG repeats, the mouse Rai1 gene contains only three CAG repeats followed by one CAA unit. This is consistent with ®ndings that the stretches of CAG repeats in other CAG-repeat containing genes are shorter in the orthologous mouse genes compared to the human genes. Examples include the human and mouse DRPLA genes, the androgen receptor (He et al., 1990), huntingtin (Lin et al., 1994) and myotonic dystrophy kinase genes (Jansen et al., 1992). Another feature in the peptide sequence is the presence of a polyserine stretch at the carboxy terminal end consisting of a [S]3A[S]8 or [S]3AA[S]3T[S]3 motif in the human and mouse peptides, respectively. Similarly, the DRPLA gene contains a non-polymorphic polyserine [S]6[A]3[S]8 motif in addition to the polymorphic polyglutamine stretch (Onodera et al., 1995). As the DRPLA gene is causally involved in a neurodegenerative disorder this points to its involvement in the development of the nervous system, and suggests an analogous role for RAI1. Another example of a gene with a polyserine domain is the Drosophila Hairless gene (Maier et al., 1992) is involved in the differentiation of neuronal cell precursors and in the development of the nervous system. Homopolimeric stretches of glutamine or proline, or proline-rich proteins, have been found to occur predomi- 74 P. Seranski et al. / Gene 270 (2001) 69±76 Fig. 3. RAI-1 expression in human tissues. Multiple-tissue northern blots from human adult tissues (a,b), brain tissues (c) and fetal tissues (d) hybridized with a 1.6 kb probe shows three different splice variants (8.0 kb, 1.5 kb, 10.0 kb, arrows). The 8.0 kb transcript is expressed ubiquitously in adult tissues (a,b) whereas it is not detected in fetal liver (d). The 1.5 kb transcript is detected only in placenta and the 10.0 kb transcript is detected in adult heart, kidney, pancreas stomach, thyroid, spinal cord and lymph node. nantly in transcription factors. There is evidence that the length of the polyglutamine and polyproline tracts directly modulate transcriptional activity (Gerber et al., 1994). This ties up with the homology to a domain of the mouse stro- melysin PDGF responsive element binding protein transcription factor (Fig. 1) and stands in contrast to the observation, that the mouse protein is localized in the cytosol of neurites and no nuclear localization signal is found. P. Seranski et al. / Gene 270 (2001) 69±76 75 3.4. Expression analysis References Northern blot hybridizations showed different splice variants of the gene: 8, 1.5 and 10 kb (Fig. 3). The 8 kb transcript could be detected ubiquitously in all tissues investigated whereas the 1.5 kb transcript was observed only in placenta. The 10 kb transcript is expressed weakly in heart, kidney, pancreas, stomach, thyroid, spinal cord and lymph node. As the 5 0 and 3 0 UTRs remain to be characterized, it is presently unclear whether the alternative transcripts arise through alternative splicing, alternative polyadenylation, or through alternative transcription initiation. Regarding the fact of the high homology between the human and mouse peptide sequences and because the differences between the human and mouse cDNA sequences are accumulated around exon boundaries, it is possible that two alternative forms of the transcript have been isolated and that RAI1 is subjected to alternative internal splicing. In order to further analyze the gene as potential Smith± Magenis candidate gene, additional expression analysis on eight different brain tissues and four fetal tissues were performed. Results showed ubiquitous expression of the 8 kb transcript of the gene in these tissues with weaker expression in cerebellum, cerebral cortex, occipital and frontal lobe. In fetal tissues, no expression was observed in liver. Mouse Rai1 is exclusively expressed in brain regions consisting predominantly of neurons like the cingulate cortex, but not in regions consisting predominantly of glial cells like the corpus callosum (Imai et al., 1995). This indicates that mouse Rai1, and by analogy human RAI1, functions in neuronal development. Further, the expression of mouse Rai1 is up-regulated by retinoic acid (Imai et al., 1995). As retinoic acid induces pluripotent P19 embryonic carcinoma cells to differentiate into neurones with the extension of neuritic processes, this underscores the functional involvement of Rai1 in neuronal development (Chiu et al., 1995). In terms of the SMS phenotype which includes craniofacial abnormalities, it is noteworthy that RA-regulated genes are involved in the development of craniofacial abnormalities as shown in RA induced asymmetric craniofacial growth in the mouse fetus (Padmanabhan and Ahmed, 1997). RAI1 therefore remains a good candidate for SMS and extensive mutation analyses are in progress to uncover putative mutations. In case of exclusion of mutations, further functional analysis of the RAI1 gene are indicated to investigate haploinsuf®ciency as a potential disease mechanism. Ban®, S., Servadio, A., Chung, M.Y., Kwiatkowski Jr, T.J., McCall, A.E., Duvick, L.A., Shen, Y., Roth, E.J., Orr, H.T., Zoghbi, H.Y., 1994. 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