Download Duplication of an approximately 1.5 Mb DNA segment

Oncogene (1997) 14, 1093 ± 1098
 1997 Stockton Press All rights reserved 0950 ± 9232/97 $12.00
Duplication of an approximately 1.5 Mb DNA segment at chromosome
5q22 indicates the locus of a new tumour gene in nonpapillary renal cell
carcinomas
Chistiane Kenck, Peter Bugert, Monica Wilhelm and Gyula Kovacs
Clinical Research Group Molecular Oncology, Department of Urology, Ruprecht-Karls-University of Heidelberg, Im Neuenheimer
Feld 365, D-69120 Heidelberg, Germany
Previous karyotyping showed an unbalanced translocation between chromosome 3p11.2-p13 and chromosome
5q22 leading to duplication of chromosome 5q22-qter
region in nonpapillary renal cell carcinomas. In order to
determine the breakpoint at chromosome 5q22 at the
molecular level, we have investigated 50 sporadic
nonpapillary renal cell carcinomas from consecutive
nephrectomies and 24 renal cell carcinomas obtained
from two patients with von Hippel ± Lindau disease. We
used seven DNA markers mapped to and around the
APC and MCC genes to detect allelic imbalance. We
observed a duplication of chromosome 5q sequences in 11
of 23 informative sporadic tumours and in 18 of 24
hereditary tumours. We determined a breakpoint cluster
between the APC and MCC genes at chromosome 5q22.
In addition we have found a partial duplication of the
smallest overlapping region of about 1.5 Mb sequences
including the MCC gene in seven tumours without visible
alteration of chromosome 5q in the karyotype. We
suggest that this DNA segment harbours a gene or gene
cluster, the altered dosage of which is important for the
growth of nonpapillary renal cell carcinomas.
Keywords: kidney cancer; chromosome 5q22; duplication
Introduction
Mutation of one allele and deletion of the wild type
allele is a well known mechanism that contributes to the
inactivation of tumour suppressor genes. The loss of
wild type alleles is indicated by gross chromosomal
deletions and by loss of constitutional heterozygosity
(LOH) detected by Southern hybridisation and polymorphic microsatellites in tumour cells. Constitutional
and/or somatic chromosomal deletions and subsequent
LOH analysis pinpointed loci of tumour suppressor
genes, and resulted in the cloning of RB, WT1, APC,
MCC, DCC and DPC4 genes. Assuming that both
deletion and duplication of chromosomes, chromosomal segments and genes can be produced by the same
genetic mechanism, such as nondisjunction and mitotic
recombination, duplications are expected to occur at the
same frequency as deletions. Trisomies or partial
trisomies of speci®c chromosomes are frequent cytogenetic ®ndings in human cancer but duplications of
chromosomal segments detected by Southern blots,
Correspondence: G Kovacs
Received 18 July 1996; revised 28 October 1996; accepted 28 October
1996
microsatellite assays or FISH were described in only a
few cases (Amler et al., 1995; Corvi et al., 1995;
Fujiwara et al., 1993; Kovacs and Kung, 1991; Tiainen
et al., 1992). A possible explanation for this controversy
is that the absence of hybridisation signal (deletion) is
easier to detect than increase of signal intensity for one
of the alleles (duplication). An increased signal intensity
might easily be misread as a LOH in tumour DNA
contaminated with normal DNA.
Comprehensive chromosome analysis has identi®ed
highly speci®c monosomies and deletions, unbalanced
translocations as well as trisomies or partial trisomies
in distinct types of renal cell tumours (Kovacs, 1993).
In nonpapillary renal cell carcinomas (RCC), which
make up about 80% of parenchymal kidney tumours,
deletions of chromosome 3p13-pter, 6q23-qter, 8p11pter, 9 and 14q22-qter regions imply the presence of
tumour suppressor genes. Loss of chromosome 3p
sequences occur in nearly 100% of sporadic nonpapillary RCCs as well as in renal tumours associated with
von Hippel ± Lindau (VHL) disease (Zbar et al., 1987;
Kovacs and Frisch, 1989; Kovacs et al., 1991). The
VHL gene, a putative tumour suppressor gene, has
already been cloned from the chromosome 3p25.3
region. Its inactivation by mutation occurs in about
50% of sporadic nonpapillary RCCs (Gnarra et al.,
1994; Kenck 1996). Cytogenetic analysis of sporadic
and hereditary nonpapillary RCCs has also revealed
trisomy or partial trisomy of chromosome 5 in about
50% of cases with the smallest overlapping region of
duplication for chromosome 5q22-qter region. An
unbalanced translocation involving the chromosomal
bands 5q22 and 3p11.2-p13 resulted in loss of
chromosome 3p and trisomy of chromosome 5q22qter sequences in several cases (Kovacs and Frisch,
1989; Kovacs et al., 1991; Presti et al., 1991). In this
paper we describe the identi®cation of a breakpoint
cluster between the APC and MCC genes at
chromosome 5q22 region in a cytogenetically wellcharacterized series of nonpapillary RCCs. We also
demonstrate that an approximate 1.5 Mb DNA
sequence distal to the critical breakpoint region is
duplicated in tumours without visible cytogenetic
alteration of chromosome 5q region.
Results
The chromosome 5q region is frequently duplicated in
nonpapillary RCCs
In order to estimate the frequency by which the
chromosome 5q region is duplicated, we have studied
Chromosome 5q22 duplication in RCC
C Kenck et al
1094
Table 1
Allelic imbalance at the chromosome 5q22 region in nonpapillary RCCs
Cytogenetic
Tumour
Allelic assignments with 5q probes
alteration of
CB86.3
FB54D
L5.71-3
YN5.48-4
MC5.61
JO205HC
Chromosome 5
D5S122
APC
MCC
D5S81
D5S372
D5S22
(a) sporadic nonpapillary RCC
295
+5q22-qter
-
12
122
-
122
122
320
+5q22-qter
12
-
-
122
-
122
344
+5q22-qter
12
-
12
12
-
-
366
+5q22-qter
12
12
122
122
112
-
400
+5q22-qter
-
-
-
122
122
-
407
+5q22-qter
12
12
-
122
122
122
284
±
12
-
112
122
12
-
314
±
12
12
112
12
-
12
319
±
-
12
-
112
-
12
324
±
-
-
122
12
12
-
359
±
12
12
122
122
12
-
379
±
12
12
112
122
-
12
391
±
-
-
-
112
-
-
(b) VHL-associated nonpapillary RCC
606
+5q22-qter
-
12
112
-
ND
112
607
+5q22-qter
-
12
122
-
ND
112
608
+5q22-qter
-
12
122
-
ND
122
623
+5q22-qter
-
12
112
-
ND
122
625
+5q22-qter
-
12
112
-
ND
122
628
+5q22-qter
-
12
122
-
ND
112
Alleles at each locus were designated 1 or 2 and refer to the larger and smaller restriction fragments, respectively. The designation 12 indicates
that the constitutional heterozygosity was retained in the karotype with an allelic dosage of 1:1, whereas 112 or 122 indicates that either allele 1
or allele 2 is present in two copies in tumour cells, as estimated from visual and densitometric analysis of autoradiographs. The entry `-'
indicates that both normal and tumour tissues were homozygous for this locus. ND, not determined
matched normal and tumour DNAs from 50 sporadic
and 24 VHL-associated nonpapillary RCCs by Southern hybridisation. A DNA probe for locus D5S22 at
the distal end of chromosome 5q was used in this assay
to detect allelic changes at critical chromosome 5q22qter segment. Twenty three out of 50 patients with
sporadic nonpapillary RCCs were heterozygous for the
locus D5S22 and 11 tumours from these patients
showed a duplication of one allele at chromosome 5q.
Twenty-four tumours obtained from two VHL patients
were included in this study. Both individuals showed
constitutional heterozygosity at locus D5S22. Eighteen
of the 24 small nonpapillary RCCs showed a
duplication for one allele at the distal region of
chromosome 5q. We did not detected deletion of
chromosome 5q sequences among 74 nonpapillary
RCCs.
Recombination breakpoint cluster between the APC and
MCC genes
Previous karyotyping localised the most common
breakpoint to the 5q22 band in nonpapillary RCCs
(Kovacs and Frisch, 1989; Kovacs et al., 1991; Presti et
al., 1991). In order to re®ne the breakpoints at the
molecular level, we have analysed the matched normal
and tumour DNAs by markers recognizing polymorphisms at the chromosome 5q22 band including those
known for the APC and MCC genes (Table 1). All
tumours with full trisomy of chromosome 5 or partial
trisomy of chromosome 5q11-qter or 5q15-qter
segments in the karyotype showed a duplication of
DNA sequences for all informative loci at the
chromosome 5q22 band (data not shown). Among
tumour samples used for this approach, six cases (295,
320, 344, 366, 400, 407) with an unbalanced
translocation and partial duplication of the chromosome 5q22-qter segment in karyotype are of particular
interest (Table 1a). The duplication of one allele of the
MCC gene and/or one allele at loci D5S81, D5S372
and D5S22 has been seen in ®ve out of six tumours.
Both alleles of the APC gene as well as of the D5S122
locus were retained in all informative cases without any
sign of allelic duplication. One of the VHL patients
with multiple bilateral tumours showed constitutional
heterozygosity at APC and MCC loci. Six out of nine
tumours showed duplication of one allele of the MCC
gene (Table 1b, and Figure 1a), whereas all tumours
retained constitutional heterozygosity without duplication of DNA sequences at the APC gene. Tumours
606, 607 and 608 were obtained from the left kidney,
tumours 623, 625 and 628 from the right kidney of one
patient. Di€erent alleles of the MCC gene were
duplicated in tumours even from the same kidney.
All six tumours showed an unbalanced translocation
3p13;5q22 in karyotype by previous cytogenetic
analysis (Kovacs et al., 1991).
Duplication of the smallest overlapping region of
approximate 1.5 Mb DNA sequences involving the MCC
gene
While searching for allelic imbalance at chromosome
5q22, we have detected a duplication of DNA
sequences in seven sporadic nonpapillary RCCs (284,
314, 319, 324, 359, 379 and 391), which by karyotyping
did not show any structural or numerical alterations of
chromosome 5 (Table 1a). An increased intensity of the
autoradiographic signal resulting in an allelic dosage of
2:1 was detected for one allele of MCC gene in each
informative case and for one allele of locus D5S81 in
®ve out of seven cases. The constitutional hetero-
Chromosome 5q22 duplication in RCC
C Kenck et al
1095
324
N
T
608
607
606
628
627
625
N
623
a
—1
—2
379
324
T
N
#3
Figure 2 Result of PCR-ampli®cation of microsatellites from the
chromosome 3p region, which demonstrates the low amount of
contaminating normal DNA in the tumour DNA samples. The
same DNA samples were used as in the Southern hybridization
experiments. The results from cases 324 and 379 are shown as
representative examples
b
N
379
N
T
T
—1
—2
YN5.48-4
—1
— 2 5.71-3
Figure 1 Representative autoradiographs showing the duplication of 5q sequences in RCCs. (a) MspI digested DNA from
normal kidney (N) and multiple tumours (623, 625, 627 and 628
from the left kidney, 606, 607 and 608 from the right kidney) of a
VHL patient was hybridized with L5.71-3 (D5S141). All but one
tumour (627) showed an allelic inbalance. Note that di€erent
alleles were duplicated even in tumours obtained from the same
kidney. (b) MspI digested DNA from normal kidney (N) and
from sporadic nonpapillary RCCs (T) with two apparently
normal chromosome 5 (cases 324 and 379) were hybridized
simultaneously with DNA probes YN5.48-4 and L5.71-3. The
constitutional heterozygosity was retained in both tumours. In
tumour 324, L5.71-3 revealed a duplication of allel 2, whereas the
allelic dosage at locus D5S81 (YN5.48-4) remained 1:1 in tumour
cells. Tumour 379 showed the duplication of one allele with both
probes. The loading of the amount of normal and tumour DNA
was controlled by ethidium bromide staining of the gels before
blotting. The degree of contamination of tumour DNA with
normal nucleic acid was controlled by ampli®cation of
microsatellites (see Figure 2)
zygosity at all other informative loci proximal (APC
and locus D5S122) and distal (loci D5S372 and D5S22)
to the duplicated segment was retained with allelic
dosage of 1:1 as seen in normal tissue. The proximal
breakpoint was localized between the APC and the
MCC gene in tumours 314, 359 and 379. The distal
breakpoint of the duplication was determined between
loci D5S81 and D5S372 in tumours 284 and 359.
Duplication of the MCC gene with retained constitutional heterozygosity and an allelic dosage of 1:1 at
locus D5S81 was seen in tumours 314 and 324,
indicating that a smaller DNA fragment is duplicated. Two examples are shown in Figure 1b. To
estimate contamination of tumour DNA with nucleic
acid from normal cells, the same DNA samples as used
in the Southern hybridization experiments were
analyzed by PCR-ampli®cation of polymorphic microsatellites from chromosome 3p region, which region is
speci®cally deleted in this type of tumour. The amount
Figure 3 Map of the chromosome 5q22 region with the
approximate locations of the probes used in this study. The
duplicated segment is indicated by a solid bar. The arrows
symbolize the locations and directions of transcription of the
APC and MCC genes
of contaminating normal DNA was low in each
tumour DNA and representative results from two
cases are shown in Figure 2. To estimate the size of the
smallest duplicated region, we used two additional
DNA polymorphisms in order to determine the distal
end of duplication in these tumours. Both DNA
probes, which recognize loci D5S1170 and D5S135
between D5S421 and D5S81, revealed a duplication of
one allele in both tumours. Therefore, the telomeric
end of the smallest overlapping duplication lies
between the loci D5S81 and D5S135 (Figure 3). The
distance between the APC gene and locus D5S81 is
estimated to be about 2 Mb. Thus, our study
determined the smallest overlapping region of duplication to be an approximate 1.5 Mb DNA segment
including the MCC gene at chromosome 5q22 in
nonpapillary RCCs.
Chromosome 5q22 duplication in RCC
C Kenck et al
1096
FISH con®rms the site of breakpoint and the duplication
of a chromosome 5q22 segment
A duplication of chromosome 5q22 sequences containing the MCC gene was detected by two-colour FISH
in interphase cells. Tumour 359 showed a karyotype 43,X,7Y,73,78,712,714,+der(8)t(8;14)(p11.2;
q11.2),+der(12)t(3; 12)(q13.2; q24.1), i.e. two apparently normal chromosomes 5 and two copies of the
long arm of chromosome 8. Two-colour FISH revealed
one, two, three and four copies of the C-MYC gene in
one, 15, one and three cells, respectively, and two,
three, four, ®ve and six copies of the MCC gene in
two, 12, one, four and one interphase nuclei,
respectively, suggesting a duplication of a chromosome 5q22 segment (Figure 4a). Tumour 391 had the
a
karyotype 46,XY,73,+7,79,+12,714,+16 without
visible structural alteration at chromosome 51. In twocolour FISH the BAC clone 83c11 harbouring the
MCC gene revealed two, three, ®ve and six signals, in
two, 13, three and two cells, respectively, whereas the
BAC clone 6e10 containing the APC gene showed two,
three, four, ®ve and six signals in three, 12, one, two
and two cells, respectively, including one weak signal
(Figure 4b). This demonstrates that DNA segments
including the MCC gene are duplicated, and also
suggest that the breakpoint might be at the telomeric
end of the BAC clone 6e10, downstream of the APC
gene.
The MCC gene is not rearranged in nonpapillary RCCs
We have localized a breakpoint cluster a chromosome
5q22 between the APC and MCC genes which bracket
an approximate 150 kb DNA segment. The two genes
are positioned head to head along chromosome 5q. As
we used the DNA probe L5.71-3, which encompasses
the ®rst two exons of the MCC gene (nt 133 ± 1918) to
detect an allelic duplication, we cannot exclude a
rearrangement within the 3' region. Therefore, we have
hybridized the 3' end of the MCC cDNA (probe MCC
40cI, nt 1634 ± 3969) to normal and tumour DNA
digested with EcoRI. We did not ®nd any di€erences in
autoradiographic signals between paired normal and
tumour samples. Therefore, a rearrangement within the
MCC gene in nonpapillary RCCs showing the t(3;5)
could be excluded with a high probability.
Discussion
b
Figure 4 (a) Two-colour FISH on an interphase nucleus of
tumour 359 shows two copies of the C-MYC (green spots) and
three copies of the MCC gene (red spots). (b) Two-colour FISH
on an interphase nucleus from tumour 391 is shown. Probe 83c11
(green spots) recognizes three copies of the MCC gene whereas
probe 6e10 (red spots) detects only two copies of the APC gene
and a small overlapping signal with probe 83c11 at the third copy
of the MCC locus
Previous cytogenetic studies showed that an unbalanced translocation between chromosomes 3q and 5q
leads to loss of chromosome 3p13-pter and duplication
of chromosome 5q22-qter sequences (Kovacs and
Frisch, 1989; Presti et al., 1991; Kovacs et al., 1991).
This cytogenetic ®nding was explained as a result of
nonhomologous mitotic recombination between chromosome 3p11.2-13 and 5q22 bands (Kovacs and Kung,
1991). We present evidence in this study that the site of
mitotic recombination at the molecular level is
restricted to an approximate 150 kb DNA fragment
between the APC and MCC genes at chromosome
5q22. We have shown in this study that the same
recombination site is involved in the duplication of the
smallest overlapping region of approximately 1.5 Mb
DNA fragment. Taking into account the results of
earlier cytogenetic analysis as well as of the present
study on the same tumour samples, the duplication of
chromosome 5q sequences occurs in about 70% of
nonpapillary RCCs. A duplication of 5q sequences was
also detected in 17% of renal cell carcinomas by
comparative genomic hybridization (Moch et al., 1996).
Strangely, not duplication but deletion of chromosome
5q22 region has been suggested by other investigators.
LOH at chromosome 5q22 has been described in 33%
of renal cell tumours analysed by Southern hybridization (Morita et al., 1991). Applying microsatellites for
allelotyping of nonpapillary RCCs, Foster et al. (1994),
Crossey et al. (1994) and Trash-Bingham et al. (1995)
found LOH at chromosome 5q each in one out of 35,
nine and 19 nonpapillary RCCs, respectively.
Chromosome 5q22 duplication in RCC
C Kenck et al
Chromosome 5q21-22 region in involved in genetic
changes of other types of cancer as well. Several studies
have demonstrated loss of one allele at chromosome
5q21-22 bands and mutation of the APC gene in the
germ line of patients with familial adenomatous
polyposis coli and also in sporadic colon cancer
(Kinzler et al., 1991; Groden et al., 1991; Nishisho et
al., 1991). Apart from colorectal cancers, frequent
LOH in the APC/MCC gene region has been found in
tumours of the liver (Fujimori et al., 1991), oesophagus
(Boynton et al., 1992) and lung (Ashton-Rickardt et
al., 1991; D'Amico et al., 1992; Fong et al., 1995).
Hosoe et al. (1994) determined the most common
region of deletions at loci D5S141 and D5S81, which is
approximately the same region found to be duplicated
in nonpapillary RCCs in this study. No mutations of
the APC and MCC genes have been detected neither in
the aforementioned tumours nor in RCCs (Horii et al.,
1992), which suggest that another tumour gene is
located in this region. The e€ect of deletion in one type
of cancer and duplication in another type is probably
dependent on gene dosage for one or more critical
genes within the 1.5 Mb region. It is also possible that
the same gene or genes are a€ected by duplication and
deletion at chromosome 5q22 in nonpapillary RCCs as
well as in the tumours of lung, liver and colon. Cloning
this gene will contribute to a better genetic characterisation of nonpapillary RCCs and some other types of
tumours as well.
Materials and methods
Tumour samples and DNA extraction
Fifty sporadic nonpapillary RCCs were obtained after
nephrectomy from the Department of Urology, at the
Medical School of Hannover. Nine nonpapillary RCCs
obtained from one patients su€ering from von Hippel ±
Lindau disease were obtained from the Department of
Urology, at the Memorial Hospital, Worchester, MA, USA
and 15 tumours from another VHL patient from the
Department of Urology, at the University of Innsbruck,
Austria. A homogeneous part of the tumour tissue was
excised immediately after nephrectomy. One third of the
specimen was used for cell culture and subsequent
chromosome analysis, one third was immediately frozen
in liquid nitrogen and stored at 7808C and the rest was
®xed in bu€ered formaldehyd for reference histology. In
the case of small tumours (2 ± 5 mm in diameter) from
VHL patients, DNA was isolated from short term cultures
of tumour cells. DNA was extracted from frozen tumour
tissue or tumour cell pellet after proteinase K digestion,
puri®ed with phenol and chloroform, precipitated with
ethanol and dissolved in TE. DNA was isolated by the
same method from normal kidney tissues.
two independent observers. DNA probes used in this
study were homologous to loci D5S122 (CB86.3), APC
(FB54D), MCC (L5.71-3), D5S1170 (f5.12), D5S135
(EF5.44), D5S81 (YN5.48-4) and D5S22 (JO205HC).
The cDNA clone FB54D includes nucleotides 6640 to
8954 of the APC gene.
Microsatellite-PCR
DNA samples used for Southern hybridization experiments
were also analysed by microsatellite-PCR with polymorphic markers (D3S1300, D3S1766) from the chromosome 3p region. PCR reactions were performed in a 20 ml
volume with 10 mmol/L Tris/HCl pH 8.3, 50 mmol/L Kcl,
1.5 mmol/L MgCl2, 0.1% bovine serum albumin,
200 mmol/L each of dATP, dCTP, dGTP and dTTP,
0.5% Tween 20, 2 pmol forward primer (5'-labelled with
1 mCi 32P), 2 pmol reverse primer, 0.5 U Taq polymerase
(Promega), and 100 ng genomic DNA as template. PCR
was carried out in a PTC100 thermal cycler (MJ Research
Inc.) with a progam as follows: initial DNA denaturation
for 2 min at 948C, 28 cycles of 948C for 40 s and 558C for
30 s and ®nal extension for 10 min at 728C. After
ampli®cation 10 ml sequencing stop solution was added
and the PCR-products were separated on 5% sequencing
gels. The dried gels were exposed to X-ray ®lms for 6 h
without using screens.
FISH-analysis of tumour cells
DNA-pools of a human BAC-library (Research Genetics)
were screened by PCR using primers for ampli®cation of
MCC (3'-end) and APC (exon 10). The BAC clones 83c11
and 6e10 were positive for MCC alone and for APC alone,
respectively. For the use in FISH-experiments the BACs
83c11 and 6e10 were labelled with biotin-11-dUTP and
digoxigenin-11-dUTP, respectively, using a nick translation
kit (Boehringer Mannheim). In another experiment the
BAC clone 83c11 was labelled with digoxigenin-11-dUTP
and cosmid clone cos-myc72, encompassing the c-myc
oncogene an chromosome 8 with biotin-11-dUTP. For
double colour FISH 60 ng probe DNA were precipitated
with 10 mg COT-1 DNA and 30 mg salmon testes DNA and
resuspended in 11 ml hybridization mixture containing 50%
formamid, 10% dextran sulphate, 26SSC, and 5 mM
sodium phosphate. Hybridization of the probes was based
on the standard Imagenetics (Naperville, Illinois) protocol
with an extended incubation time over 3 days at 378C. The
slides were then incubated ®rst with avidin-FITC (10mg /
ml) for 30 min, then with biotinylated goat anti-avidin
(5 mg/ml) and anti-digoxygenin-rhodamin (10 mg/ml) for
75 min and ®nally with avidin-FITC (10 mg/ml) for 30 min,
each step at 378C. The nuclei were stained with 50 ng/ml
DAPI for 8 min and mounted with 30 ml antifade solution.
The results were visualized under a Leitz DMRBE
¯uorescens microscope equipped with a Photometrics
NU200 CCD camera using IPLab Imagining Processing
and Multiprobe software. Twenty metaphases were
counted for hybridizations signals.
Southern-blotting, hybridisation and DNA probes
Eight mg of normal and tumour DNA were digested with
appropriate restriction enzymes according to the manufacturer's instructions (Boehringer Mannheim) and fractionated by electrophoresis on 0.8 ± 1.0% agarose gels. After
denaturation and neutralisation DNA was transferred to a
charged nylon membrane (Hybond N+, Amersham). The
DNA probes were labeled with [a 32P]dCTP by random
primer extension. After prehybridization ®lters were
hybridized with the labelled probes in the same solution.
Autoradiographs were assessed for allelic imbalance by
Acknowledgements
This work was supported by the German Research Council
and in part by the EMBO (short term fellowship to GK).
The authors thank Sir Walter Bodmer for his support. The
cDNA probes FB54D, MCC 40cI were kindly provided by
Bert Vogelstein (The Johns Hopkins University), the
L5.71-3, EF5.44, YN5.48-4 and MC5.61 probes by
Yusuke Nakamura (Tokyo University) and the probe
f5.12 by Lynn Jorde (University of Utah Health Sciences
Center).
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Chromosome 5q22 duplication in RCC
C Kenck et al
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