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).
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expression of mouse Rai1 is up-regulated by retinoic acid
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candidate for SMS and extensive mutation analyses are in
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Acknowledgements
We appreciate the skilful technical assistance of Esther
Backes, Tanja Detzel and Lillia Holmes. This project was
funded in part by the BMBF grant No. 01KW9704/0 and a
grant from the South Carolina Department of Disabilities
and Special Needs (SCDDSN).
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