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Cytogenet Cell Genet 73:179-183 (1996)
Cytogenetics and
Cell Genetics
sarcomas
B. de Leeuw, M. Balemans, and A. Geurts van Kessel
Department of Human Genetics, University Hospital, Nijmegen (The Netherlands)
Abstract. The human synovial sarcoma-specific transloca
tion t(X; 18) results in the fusion of the SYT gene on chromo
some 18 with either one of the Kruppel-associated box (KRAB)
containing SSX1 or SSX2 genes on the X chromosome, de
pending on the exact location of the breakpoint within band
X p ll.2 . Screening of a testis cDNA library yielded several
SSX-positive clones. Subsequent sequence analysis revealed
that one third of these clones represent an SSX gene that differs
from both SSX1 and SSX2. This novel member of the family of
Synovial sarcomas are soft-tissue tumors that occur mainly
in adolescents and young adults. The chromosomal transloca
tion t(X; 18)(pl 1.2;ql 1.2) is found in the majority of these sar
comas and, as such, is thought to play a causative role in tumor
formation (Turc-Carel et al., 1987). Two distinct chimeric
products have been identified in different t(X; 18)-positive
synovial sarcomas, resulting from the fusion of the SYT gene
on chromosome 18 to either the SSX1 or the SSX2 gene on the
X chromosome (Clark et al., 1994; Leeuw et al., 1994a, 1995;
Crew et al., 1995). These alternative fusion products have been
correlated with different X-chromosomal breakpoints in fluo
rescence in situ hybridization experiments using Xp 11.2-spe
cific YACs as probes (Leeuw et al., 1993a, b, 1994b; Olde
Weghuis et al., 1994; Shipley et al., 1994; Janz et al., 1995).
Interestingly, there appears to be a positive relationship be
tween the occurrence of these alternative X-chromosomal
breakpoints and the most predominant histologic characteris
tics of the tumors, namely, whether they are monophasic or
Supported by the Dutch Cancer Society (Koningin Wilhelmina Fonds).
Received 1 November 1995; accepted 15 January 1996.
Request reprints from Dr. A. Geurts van Kessel, Department of Human Genetics,
University Hospital Nijmegen, PO Box 9101, 6500 HB Nijmegen
(The Netherlands); telephone: 31-24-3614107; fax: 31-24-3542151.
YCA R C\ F R
l\/* V I \U 1 1 \
E-mail [email protected]
Fax+ 41 61 306 12 34
http:// www. karger, ch
© 1996 S. Karger AG, Basel
0301-0171/96/0733-0179$ 10.00/0
KRAB containing SSX genes, which we designated SSX3, is
90% homologous to SSX1 and 95 % homologous to SSX2 at the
cDNA level. Somatic cell hybrid analysis indicated that SSX3
maps within Xpl 1.2-»p i 1.1, the region that also harbors the
SSX1 and SSX2 genes. However, we conclude from our RTPCR data and from results reported in the literature that SSX3
does not act as a fusion partner to SYT in any of the 44 inde
pendent synovial sarcomas thus far tested.
biphasic (Leeuw et al., 1994b; Janz et al., 1995). Here, we
report the identification and chromosomal localization of a
third member of the family of Kruppel-associated box contain
ing SSX genes. This gene, however, is not implicated in t(X; 18)positive synovial sarcomas.
Materials and methods
Library screening and sequence analysis
The human fibrosarcoma HT1080 (Clonetech) and the human testis 5'
stretch (Clonetech) cDNA libraries were used for screening, using essentially
the same standard procedures described as before (Leeuw et al., 1993b,
1994a). DNA sequences were analyzed on an automated DNA sequencer
(ABI 373A) using a Taq dye deoxy terminator cycle sequencing kit (Applied
Biosystems).
Patient material and RT-PCR analysis
The patient material used in this study included nine synovial sarcomas
that were extensively analyzed before (Leeuw et al., 1995) and six novel
tumors. Diagnosis of these latter six tumors as synovial sarcomas was con
firmed via a positive SYT-SSX RT-PCR score. RT-PCR and subsequent
restriction enzyme analyses were performed as described previously (Leeuw
etal., 1995).
Southern blot analysis
DNAs of hybrid and parental cell lines were isolated as described before
(Leeuw et al., 1994a); digested to completion with a variety of restriction
endonucleases (Life Technologies); and, after agarose gel electrophoresis,
SSX1
SSX2
SSX3
ATGAACGGAG ACGACACCTT TGCAAAGAGA CCCAGGGATG
ATGAACGGAG ACGACGCCTT TGCAAGGAGA CCCACGGTTG
ATGAACGGAG ATGACACCTT TGCAAGGAGA CCCACGGTTG
SSXl
SSX2
SSX3
AATGATTCGA AGGGAGTGTC AGAAGCATCT GGCCCACAAA
AATGATTCGG AGGAAGTGCC AGAAGCATCT GGCCCACCAA
AATGTTTCGA AGGAAGTGCC AGAAGCATCT GGCCCACAAA
Lspl
SSX1
SSX2
SSX3
ATGCTAAAGC ATCAGAGAAG AGAAGCAAGG CCTTTGATGA
GTGCTCAAAT ACCAGAGAAG ATCCAAAAGG CCTTCGATGA
GTGCTCAAAT ACCAGAGAAG ATACAAAAGG CCTTCGATGA
SSXl
SSX2
SSX3
ACGATGGGAA ACAACTGCAC CCCCCAGGAA AAGCAAATAT
AT GAT GGGAA AGAGCTGTGC CCCCCGGGAA AACCAACTAC
ACGATGGGAA ACAGCTGTGC CCCCCGGGAA AACCAACTAC
Sma I
SSX1
SSX2
SSX3
TATTGCCACA TACTTCTCTA AGAAAGAGTG GAAAAAGATG
TATTGCCAAA TACTTCTCTA AGGAAGAGTG GGAAAAGATG
TATTGCCAAA TACTTCTCTA AGGAAGAGTG GGAAAAGATG
SSXl
SSX2
SSX3
TTCTGAGAAG ATTAATAAGA GATCTGGACC CAAAAGGGGG
CTCTGAGAAG ATTCACGAGA GATCTGGACC CAAAAGGGGG
CTCTGAGAAG ATTAACATGA TATCTGGACC CAAAAGGGGG
E c o R V / B g lX I
SSX1
SSX2
SSX3
AAATACTCAG AGAAAATCAG CTATGTGTAT ATGAAGAGAA
AAAGCCTCAG AGAAAATCTT CTATGTGTAT ATGAAGAGAA
AAAGTCTCGG AGAAAATCGT CTATGTGTAT ATGAAGAGAA
SSXl
SSX2
SSX3
AAACATGCCT GGACCCACAG ACTGCGTGAG AGAAAGCAGC
GAACATGCCT GGACCCACAG ATTGCGTGAG AGAAAACAGC
GAACATGCCT GGACCCACAG ACTGCGTGAG AGAAAACAGC
SSX1
SSX2
SSX3
ACTATAAGGC CATGACTAAA CTAGGTTTCA AAGTCACCCT
AGTATGAGGC TATGACTAAA CTAGGTTTCA AGGCCACCCT
AGTATGAGGC CATGACTAAA CTAGGTTTCA AGGCCATCCT
SSXl
SSX2
SSX3
TGGTGATTTA TGAAGAGATC AGCGACCCTG AGGAAGATGA
TGGTGATTTA TGAAGAGATC AGCGACCCTG AGGAAGATGA
TGGTGATTTA TGAAGAGATC AGCGATCCTG AGGAAGATGA
SSX1
SSX2
SSX3
CCCACCTTTC ATGTGTAATA AACAGGCCAC AGACTTCCAG
CCCACCTTTC ATGTGTAATA TACGGGCCGA AGACTTCCAG
CCCATCTTTC ATGCGTAATA AACGGGTCAC AGACTTCCAG
SSXl
SSX2
SSX3
CGAGTAACTC CCCTGGGGGA TACGACACAT GCCCTTGATG
CGACTAACTC CCCTCAGGGA TACGACACAT GCCCATGATG
TGAGTAACTC CCCTTGGGGA TATGACACAT GCCCATGATG
SSX1
SSX2
SSX3
GGGAATGATT TTGATAATGA CCATAACCGC AGGATTCAGG
GGGAATGATT TGGATAATGA CCCTAACCGT GGGAATCAGG
GGGAATGATT TTGATAATGA CCCTAACCGT GGGAATCAGG
SSXl
SSX2
SSX3
AGAAGCAGAA CGTGGTGACC TTTCACGAAC ATGGGCATGG
AGAAGCAGAA CGTGGTGACC TTTCACGAAC ATGGGCATGG
AGAAGCAGAA CGTGGTGACC TTTCACGAAC ATGGGCATGG
SSX1
SSX2
SSX3
TTGAACATCC TCAGATGACT TTCGGCAGGC TCCACAGAAT
TTGAACGTCC TCAGATGACT TTCGGCAGGC TCCAGGGAAT
TTCTACGTCC TCAGATGACT TTCGGCAGGC TCCAGGGAAT
SSXl
SSX2
SSX3
CTGCGGCTCC CTCGTCATCA GGTGCATAGC AAGTG
CTGCGGCTCC CTCGTCATCA GGTGCATAGC AAGTG
CTGTGGACCC CTCGTCATCA GGTGCATAGC AAGTG
SSX1
SSX2
SSX3
CATCCCGAAG ATCATGCCCA AGAAGCCAGC AGAGGACGAA
CTCCCCGAAG ATCATGCCCA AGAAGCCAGC AGAGGAAGGA
CTTCCCGAAG ATCATGCCCA AGAAGCCAGC AGAGGAAGGA
Lt r a n s l o c a t i o n b r e a k p o i n t
Fig. 1. The SSX3 cDNA sequence compared to those of SSX1
Bglll, EcoRV, Smal, and Lspl restriction sites are indicated.
M 1 2 3 4 5
Fig. 2. Restriction digests of SSX3 testis cDNA clones (BamHl-Xbal
inserts into pDR2) using BamHU Bglll (lanes l—5) or BamHl/IuvR'V (lanes
6-10). Lanes 1 and 6: pDR2-30; lanes 2 and 7: pDR2-33; lanes 3 and 8:
pDR2-34; lanes 4 and 9: pDR2-36; lanes 5 and 10: pDR2-42. Lane M: size
marker (100-bp ladder, Life Technologies). The 6()0-bp marker band is indi
cated by an arrow. The asterisks indicate a 1.3-kb Baml-l if EcoRV vector
blotted onto Genescreen Plus membranes (Dupont) using standard protocols. Blots were hybridized overnight in 0.5 mM PBS, 1 mM NaaEDTA, and
7% (h’/ vJ SDS at 65 °C; washed once in 40 mM phosphate, 0.1 % SDS and
then in 10 mM phosphate, 0.1 % SDS at 650 C; and, lastly, exposed to X-ray
film (Kodak) at - 8 0 ° C for 1-3 d, using intensifying screens.
Results and discussion
Screening of a hum an fibrosarcoma cDNA library with a
synovial sarcoma-derived SYT-SSX RT-PCR product (Leeuw
et al., 1995) yielded several partial SSX2 fragments. One of
180
Cytogcnet Cell Genet 73:179-183 (1996)
6 7 8 9 10
M
*
these fragments was used as a probe to screen a human testis
cDNA library, which resulted in 15 positive clones. Five of
these full-length clones (pDR2-30, pDR2-33, pDR2-34, pDR236, and pDR2-42) were found to lack a Bglll site that is normal
ly present in both the SSXl and SSX2 cDNAs (Leeuw et al.,
1995). Sequencing of these five clones yielded identical results
and revealed a novel SSX transcript that was significantly dif
ferent from SSXl and SSX2 (for comparison, see Fig. 1). Overall, this novel sequence, which we designated SSX3, is 90%
homologous to SSXl and 95% homologous to SSX2 at the
cDNA level. At the position of the Bglll site in SSXl and SSX2
M
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Fig. 4. Amino acid sequences of SSXl, SSX2, and SSX3
and 3, respectively). Differences as compared to the SSX2 sc
cated by asterisks. The KRAB boxes are marked with the conserved amino
acids in boldface type, and the semiconserved amino acids are underlined
(modified after Crew et al., 1995).
SÄiiiiiSf
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FI*. 3. R estric tio n <liecst „fS Y T -ssx R T - P C R p ro d u cts from ! 2 d iiT Crent synovial sarcomas with EcoRV (A), Bglll (B), Lspl (C), and Smal (D). The
PCR was performed on 1 jxl of RT material with SYT (5'-CAACAGCAAGATGCATACCA-3') and SSX (5'-CAClTGCTATGCACCTGATG-3')
primers (Leeuw et al., 1995), I min at 92°C, 1 min at 48°C, and 3 min at
72“C for 35 cycles. Lanes 1-12 contain RT-PCR material from tumors 9450418, 89-52115, 2374/90, CATC, 88-50654, 243090, Hiss, KN, PTN SSI,
4873/92, 950501XC, and 2214-287, respectively (Leeuw et al., 1993a,
1993b, 1994a, 1994b, 1995; Janz et al., 1995; and unpublished cases). Lane
M: size marker (100-bp ladder, Life Technologies). The 600-bp marker band
is indicated by an arrow.
(Figs. I and 2) an EcoRV site is found in SSX3 that is not
present in SSX 1 and SSX2. Aside from these differences, SSX3
shows the same Smal site as SSX2 (absent from SSXl) and the
same Lspl site as SSX 1 (absent from SSX2). As a consequence,
digestion of SYT-SSX RT-PCR products must reveal Bglll and
Lspl sites in case SSXl is involved, Bglll and Smal sites in case
SSX2 is involved, and Smal, Lsph and EcoRV sites in case
digestion of SYT-SSX RT-PCR products from nine different
synovial sarcomas revealed fusion products that contained only
SSX 1- or SSX2-derived sequences (Leeuw et al., 1995). Digestion of RT-PCR products from six additional independent
synovial sarcomas revealed a Bglll site and either an Sma\ or
Lspl site, whereas none showed an EcoRV site or both ¿7««I
and Lspl sites (Fig. 3). From these results we conclude that the
SSX3 gene is not fused to SYT in any of our 15 independent
synovial sarcomas.
C r e w c t a1' (1 9 9 5 ) P “ 1*
"
a series o f 2 9 sy n o v ia l s a r c o m a s
?1f t\
SSX RT-PCR products, but never both sites
tioned that only SSXl or SSX2 sequences were encountered.
Therefore, we conclude that SSX3 is not included in the fusion
with SYT in any of the 44 independent synovial sarcomas thus
far tested. In addition, three synovial sarcomas without any evideuce for the presence of SYT-SSX1 or SYT-SSX2 fusion products were
ted (Crew et al.,
in these cases
with certainty, but it
would h
detected by the investigators using the highly
Figure4 compares the amino acid sequences of SSXl,
SSX2, and SSX3. It can be deduced from this comparison that,
at this level, the identity between SSX2 and SSX3 is relatively
high (90 %), except for the Kruppel-assoeiated box B (KRAB B)
region, where it is only 73%. The overall identity between
SSXl and SSX2 is only 76%. Whereas the KRAB A domain
appears to be involved in the repression of transcription,
B domain seems to be dispensable f
activity under in vitro conditions ( Witzgall et al., 1994). It
been suggested that KRAB-eontaining proteins may exert their
effects through interactions with transcriptional activators containing a glutamine-rich activation domain (Licht et al., 1993).
« j jt M * % í is a t3 * « íia M N s # * i» w s 5 T W * y « w t f 4 » * ! tiA U « ^ iP i ¿ ^ * > r v y t^ X r» » u ¿ tA ^ iy M $ * n i.?ía
Cytogenet Cell Genet 73:179-183(1996)
181
A
B
C
D
E
Fig. 5. Southern blot analysis of
¿scoRI-digested somatic cell hybrid
(578, 578K17, Hlsynsarc; lanes C,
D, and E, respectively) and control
Chinese hamster (A3, lane A) and
human (HL60, lane B) cell line
DNAs. Size markers are deduced
from a co-electrophoresed 1-kb lad
der (BRL).
As such, the observed differences at the amino acid level
between the KRAB B domains of SSX2 and SSX3 may give rise
to functionally different interactions. The exact nature of these
putative interactions, however, remains to be established.
The chromosome location of the SSX3 gene was established
through the analysis of a panel of human x rodent somatic cell
hybrids including the X-only line 578 (Sinke et a l, 1993); its
radiation-reduced derivative 578K17, which contains
Xp 11.4 -» p 11.1 segment as the only human constituent (Berger
et al., 1992); and the synovial sarcoma-derived line Hlsynsarc
containing X p ll.2 - > q te r (Leeuw et al., 1993a). Since 578,
578K17, and Hlsynsarc exhibit restricion fragments similar to
in control total human genomic DNA that
hybridizes to our SSX3 cDNA probe (absent in the control
hamster DNA under the stringency conditions applied [Fig. 5]),
we conclude that the SSX3 gene must reside within X pl 1.2
p 11.1. This proximal Xp segment contains several low-copy
repeats, among which are the OAT-like sequences (Sinke et al.,
1993; Leeuw et al., 1994b). Interestingly, SSX1 is positioned
precisely within the OATL1 cluster (Leeuw et al., 1993a). The
identification of yet another SSX gene (SSX3) in X p ll.2 ->
p i 1.1 is in full agreement with the repeated nature of this par
ticular chromosomal segment (Lafreniere et al., 1991). Since
SSX1 and SSX2 are functional, expressed genes containing
intronic sequences (unpublished data), they do not seem to
182
Cytogenet Cell Genet 73:179-183 (1996)
have arisen by reversed transcription. In this respect, they are
the interspersed OAT-like sedifferent from, for
quences, some of which have been reported to be processed
pseudogenes (Lafreniere et al., 1991; Geraghty et a l, 1993). It
has been hypothesized before that the OATL1 and OATL2
clusters may have evolved via a duplication event of the entire
region (Shipley et al., 1994). Our present results indicate that
during the course of evolution, several of these duplication
events must have occurred within this particular genomic seg
ment.
In addition to the SSX3 gene reported here, we have evi
dence for the existence of at least two other SSX-like genes. The
first one, designated SSX4, was found after RT-PCR with two
SSX internal primers oil RNA extracted from a primary human
fibrosarcoma. Preliminary sequence data indicate that this
gene may give rise to a protein truncated just after the KRAB A
box. The second one, SSX5, was detected via the presence of an
exon in one of our OATL1 YAC-derived cosmids (Leeuw et al.,
1993b). This exon shows between 80% and 90% base-pair
homology to the corresponding exons in SSX1, SSX2, SSX3,
and SSX4. Whether SSX4 and SSX5 actually represent func
tional genes still remains to be established. The possible
involvement of any of these novel SSX sequences in the devel
opment of other neoplastic disorders carrying X-autosome
translocations, such as renal cell carcinomas (Sinke et al., 1993;
Dijkhuizen et al., 1995), is currently under investigation.
Acknowledgements
The authors thank C. Ture-Carel, G. Stcnman, L.G. Kindblom, N. Mandahl, S. Gilgenkrantz, J.M. Lopes, J.A. Fletcher, W.M. Molenaar, and T.
Wobbes for providing tumor material.
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