Download - Aberrant splicing of the PTPRD gene mimics microdeletions identified at this locus in neuroblastomas
Aberrant Splicing of the PTPRD Gene MimicsMicrodeletions Identified at this Locusin Neuroblastomas
Prakash Nair,1 Katleen DePreter,2 Jo Vandesompele,2 Frank Speleman,2 and Raymond L. Stallings1*
1Departmentof Pediatrics,Greehey Children’s Cancer Research Institute,The Universityof TexasHealth Science Center at San Antonio,San Antonio,TX 782292Center for Medical Genetics,Ghent University Hospital,Ghent,Belgium
Neuroblastoma (NBL), a pediatric tumor arising from precursor cells of the sympathetic nervous system, is characterized by
numerous recurrent large-scale chromosomal imbalances. High resolution oligonucleotide array CGH analysis of NBL has pre-
viously identified microdeletions that are confined to the 50 UTR of the protein tyrosine phosphatase receptor D (PTPRD)
gene, implicating this gene in the pathogenesis of these tumors. Here, we demonstrate that the 50 UTR of this gene, consisting
of 11 noncoding exons, is also aberrantly spliced in >50% of NBL primary tumors and cell lines. The loss of exons from the 50
UTR region through aberrant splicing results in aberrant mRNA isoforms that are similar to those generated through microde-
letions. The aberrant splicing or microdeletion of 50 UTR exons in such a high proportion of tumors indicates that loss of these
exons dys-regulates the mRNA sequence. To further validate the role of PTPRD in NBL, we have examined the expression of
this gene in normal fetal adrenal neuroblasts (the cell of origin of NBL) and in tumors from patients with either low stage or
high stage disease. This gene is expressed at lower levels in high stage NBL tumors, particularly those with amplification of
MYCN, relative to low stage tumors or normal fetal adrenal neuroblasts, consistent with the possibility that loss of the 50 UTRexons have destabilized the mRNA. This article contains Supplementary Material available at http://www.interscience.wiley.
com/jpages/1045-2257/suppmat. VVC 2007 Wiley-Liss, Inc.
INTRODUCTION
Neuroblastoma (NBL), a pediatric cancer
derived from primitive cells of the sympathetic
nervous system (Brodeur, 2003), can be subdivided
into at least three genetic subtypes with dramati-
cally different clinical behaviors, those with MYCNamplification, 11q loss, or near triploid (Vandesom-
pele et al., 1998). The near triploid tumors gener-
ally have favorable clinical outcomes, while tumors
with either MYCN amplification or 11q loss often
become refractory to treatment (Brodeur et al.,
2001).
Hemi- and homozygous microdeletions affecting
either the 50 UTR or coding sequence regions of
the protein tyrosine phosphatase receptor D
(PTPRD) gene have been reported in NBL (Stal-
lings et al., 2006). In addition, expression of
PTPRD is higher in neuroblasts that were microdis-
sected from normal fetal adrenal gland than in tu-
mor material derived from patients with Stage 4
disease (De Preter et al., 2006). These normal neu-
roblasts have been shown to be the cell of origin of
NBL. Interestingly, PTPRD appears to play a wide-
spread role in cancer, as microdeletions (Sato et al.,
2005; Zhao et al., 2005; Purdie et al., 2007; Stark
and Hayward, 2007) or mutations (Sjoblom et al.,
2006) affecting this gene have been detected in
several other forms of cancer. PTPRD protein
interacts with a putative metastasis suppressor,
MIM, which is involved with cytoskeletal remodel-
ing (Woodings et al., 2003; Gonzalez-Quevedo
et al., 2005).
The genomic architecture of PTPRD is complex,
with 36 coding sequence exons and a 50 UTR
spliced together from 11 noncoding exons. Multi-
ple isoforms are generated by either alternate splic-
ing (Pulido et al., 1995) or by alternate transcrip-
tional start sites (Sato et al., 2005) in a tissue spe-
cific manner. The predominant isoform in brain
has an extended 711 base pair 50 UTR (L isoform),
while the isoform (S) expressed in kidney lacks the
extended 50 UTR (Sato et al., 2005). In addition,
the brain isoform is characterized by the absence
*Correspondence to: Raymond L. Stallings, Royal College of Sur-geons in Ireland, 123 St Stephen’s Green, York House, Dublin 2,Ireland or Children’s Medical and Research Foundation, Our Lady’sHospital for Sick Children, Dublin 12, Ireland.E-mail: [email protected]
Supported by: Association of International Cancer Research.
Received 28 September 2007; Accepted 26 October 2007
DOI 10.1002/gcc.20521
Published online 29 November 2007 inWiley InterScience (www.interscience.wiley.com).
VVC 2007 Wiley-Liss, Inc.
GENES, CHROMOSOMES & CANCER 47:197–202 (2008)
of exons 14 to 18 corresponding to amino acid resi-
dues 568 to 978 of the 4th through 7th fibronectin
III-like domain and by the insertion of a 12 base
pair mini-exon sequence between exons 23 and 24
(Pulido et al., 1995).
In spite of the increasing evidence for the role of
PTPRD in tumor pathogenesis, relatively little is
understood about the expression and splicing of
this gene in tumors. It was demonstrated that lung
carcinoma cell lines with PTPRD deletions have
lower levels of mRNA transcripts relative to nor-
mal lung tissue (Sato et al., 2005). In addition, Pur-
die et al. (2007) have shown that an intragenic de-
letion of PTPRD identified in a squamous cell car-
cinoma created a fusion allele consisting of exon
B8 of the 50 UTR fused to coding sequence exon
5, which is expressed in the tumor cells, perhaps
conveying a dominant negative effect. Here, we
examine both the expression and splicing of
PTPRD in primary NBL tumors and cell lines.
MATERIAL ANDMETHODS
Cell Lines and Tissues
Protein tyrosine phosphatase receptor D
(PTPRD) studies were performed on primary NBL
tumor tissues obtained from a tumor bank at Our
Lady’s Hospital for Sick Children (Dublin, Ire-
land) and on cell lines obtained from either the
American Type Culture Collection or the Child-
ren’s Hospital of Los Angeles. The International
Neuroblastoma Staging System was used to desig-
nate disease stage (Shimada et al., 1999). Basically,
Stage 1 or 2 disease involves local-regional tumors,
where metastasis (if any) is limited to nearby
lymph nodes, while Stage 4 involves metastasis to
distant organ sites.
Affymetrix HU-133a Expression Microarray
Analysis of Microdissected Fetal Neuroblasts
and Primary Tumors
Details of the characterization of microdissected
normal fetal adrenal neuroblasts, including micro-
array expression analysis, has been provided in
detail by (De Preter et al., 2006).
Quantitative Real Time PCR and PCR Analysis
of mRNA Splicing
All primers used for quantitative real time PCR
analysis of either cDNA or genomic DNA are
listed in Supplementary Table 1. Expression of
PTPRD was quantified using two-step reverse tran-
scriptase quantitative PCR analysis. The primers
for real time PCR analysis were derived from exon
B1 (50 UTR)(forward primer) and exon 1 (coding
sequence)(reverse primer). Both of these exons
were found to be consistently expressed in all
tumors and cell lines that were analyzed for
mRNA splicing.
Primers used in the analysis of mRNA splicing
were designed to specifically amplify exon regions
B2, B4, B5, B7, B8, B10, B11, E2, or E3 (forward
primers) to exon E5 (reverse primer) from cDNA.
GAPDH was used to normalize the expression lev-
els in each sample. cDNA was first synthesized
from total RNA using oligo dT primers and the
TaqMan Reverse Transcriptase (RT) kit from
Applied Biosystem (Foster City, CA). Hundred
and fifty nanogram of total RNA was used for
cDNA synthesis in a total reaction volume of 30 ll,which included the 1X RT buffer, 5.5 mM MgCl2,
500 lM dNTP, 2.5 lM oligo dT, 0.4 U/ll of RNase
inhibitor, and 1.25 U/ll Multiscribe Reverse Tran-
scriptase. The cycling parameters were 258C for
10min, 488C for 30min, and 958C for 5min. The syn-
thesized cDNA was used for the real-time Q-PCR
reaction on an ABI 7500 using triplicate runs per
sample-primer pair. Each Q-PCR reaction (20 ll)contained 2 ll of cDNA, 0.2 lM each of forward
and reverse primers, and 1X SYBR green master
mix. The cycling condition was 1 cycle each of
2 min at 508C, and denaturation at 958C for 10 min,
followed by 40 cycles of 958C for 15 sec and 608Cfor 60 sec. The relative quantitation assay type was
used for experimental setup and the data was ana-
TABLE 1. PTPRD 50 UTR Exon Expression
Sample Stage 50 UTR exons not expresseda
T-39 4 B2, B4, B5, B7, B10b, B11c
T-53 DUB 2T-31 4T27 2aT1 2IMR32Kelly B7, B10CHP212SK-N-FI B2, B4, B7b
SK-N-DZ B2, B4, B5, B7, B8, B11, E3b
SMS-LHNSHSY5Y B2, B4, B5, B7, B8, B10NGP B2, B4, B7, B11SK-N-BE B2, B4, B5, B7, B8, B11NB69LA-N-6 B2c, B4, B5, B7, B8, B10SK-N-AS B2, B4, B5b, B7, B8c, B11c
aExons B2, B4, B5, B7, B8, B10, B11 from the 50 UTR and E2 and E3
from the 50 region of the coding sequence were assayed.bFaint band.cAbnormal size.
Genes, Chromosomes & Cancer DOI 10.1002/gcc
198 NAIR ET AL.
lyzed using the comparative Ct (DDCt) method.
The relative expression levels (compared with
total RNA from brain tissues) were calculated from
the respective Ct value as 22DDCt, where 2DDCtrepresents the ratio of the normalized expression
in sample and the control brain tissue. The relative
expression 6 standard error is plotted for each
sample. Real timer PCR analysis of genomic DNA
was carried out as previously described (Selzer
et al., 2005).
RESULTS
PTPRD Expression in Neuroblastoma
De Preter et al. (2006) have recently determined
that fetal adrenal neuroblasts are the cell of origin
of neuroblastomas (NBLs) and have examined the
expression profiles of microdissected normal fetal
adrenal neuroblasts and different genetic subtypes
of primary NBL using Affymetrix HU-133A arrays
(De Preter et al., 2006). A review of the primary
data from this study shows that PTPRD is
expressed at higher levels in both normal fetal ad-
renal neuroblasts and loco-regional tumors relative
to MYCN amplified or 11q- tumors derived from
patients with high stage disease (i.e., metastatic
disease) (Fig. 1). No information regarding the
expression of the L isoform (containing the 50
UTR) in NBL was obtainable from this study since
only the extreme 30 region of the PTPRD gene is
represented on these arrays. Therefore, we have
carried out expression analysis of PTPRD on 29 pri-
mary NBL tumors representing low stage near trip-
loid tumors with favorable histopathology (n 5 9),
high stage 11q- tumors with unfavorable histopa-
thology (n 5 10) and high stage MYCN amplified
tumors with unfavorable histopathology (n 5 10)
using quantitative real time PCR (Fig. 2). Previous
analysis of 15 of these tumors by array CGH indi-
cated that none of these tumors had detectable de-
letion of the PTPRD gene (Stallings et al., 2006).
PTPRD deletion status of the other 14 tumors is
unknown. Primers specific for the L isoform, as
originally described by Sato et al. (2005), came
from exon B1 (forward primer)(exons designated B
are from the 50 UTR) and from exon 1 (reverse pri-
mer)(see supplementary information on PCR pri-
mers). The L isoform was expressed in NBL, simi-
lar to brain tissue. Consistent with the microarray
study (De Preter et al., 2006), the mean expression
in low stage hyperdiploid tumors (mean 5 0.7) was
greater than either the 11q- (mean 5 0.3) or MYCNamplified (mean 5 0.07) tumors. The difference in
mean expression levels between the three tumor
groups was statistically significant using the non-
parametric Kruskal-Wallis test (P < 0.001).
PTPRD mRNA Splicing in Neuroblastoma
To determine if NBL have the PTPRD splice
variant that is predominant in brain (i.e., lacking
exons 14 to 18), we sequenced PCR products
obtained from cDNA from 12 tumors using a for-
ward primer from E11 and a reverse primer from
E28. All 12 tumors, which included five 11q-
tumors, two MNA tumors and five low stage hyper-
diploid tumors, lacked exons 14 through 18, similar
to the brain isoform. All of these tumors also con-
tained a mini-exon sequence (TAAACCCGACAG)
between exons 23 and 24, similar to the brain
isoform.
Figure 1. PTPRD expression in normal fetal neuroblasts, low stagehyperdiploid neuroblastomas with favorable prognosis, high stage neu-roblastomas with loss of 11q and unfavorable prognosis, and high stageneuroblastomas with MYCN amplification and unfavorable prognosis.PTPRD expression data from Affymetrix HU-133a microarrays wasobtained from De Preter et al. (2006).
Figure 2. PTPRD expression in high stage neuroblastomas withMYCN amplification, high stage tumors with loss of 11q, and low stagenear triploid neuroblastomas. PTPRD expression was ascertained usingquantitative real time PCR. The primers for real time PCR analysiswere derived from exon B1 (50 UTR) and exon 1 (coding sequence).Both of these exons were found to be consistently expressed in alltumors and cell lines that were subsequently analyzed for mRNA splic-ing (Table 1 and Fig. 4).
Genes, Chromosomes & Cancer DOI 10.1002/gcc
199ABERRANT SPLICING OF PTPRD IN NEUROBLASTOMA
In order to further examine the splicing of the 50
region of PTPRD in NBL tumors and cell lines, we
have carried out PCR analysis on cDNAs using for-
ward primers from exons B2, B4, B5, B7, B8, B10,
B11, exon 2 and exon 3 individually combined
with a reverse primer from exon 5 (see supplemen-
tal section for primer sequences). Figure 3a illus-
trates the expected pattern of the PCR products
from brain cDNA (expected to have product from
all primers). As expected, the PCR products
decrease in size going from exons B2 to E3 (Fig.
3a). PCR products using primers from the 50 UTR
exons B2 to B11 in combination with a reverse
primer from exon 5 were absent from kidney
cDNA, as expected (Fig. 3b). Only forward primers
from exons 2 and 3 produced PCR product from
cDNA derived from kidney total mRNA (Fig. 3b),
consistent with a published study indicating that
kidney expresses only the S isoform (Sato et al.,
2005). Analysis of cDNA from five primary tumors
and 12 cell lines indicated that exons B2 through
exon 3 were present in eight samples (Fig. 4a).
However, in nine samples, including both primary
tumors and cell lines, various exons from the 50
UTR were not present in cDNA, as illustrated in
Figures 4b–4d and summarized in Table 1. Exons
B2 and B4 were the most frequently missing 50
UTR exons. In some instances, PCR products
were not of the expected size (Table 1), which
might also be indicative of splicing aberrations.
The Kelly and SK-N-AS cell lines had homozy-
gous deletion of the PTPRD 50 UTR region, as
indicated by array CGH and real time PCR analy-
sis of genomic DNA (Stallings et al., 2006), so that
absence of some of these exons in cDNA would be
expected. The presence of exon B8 and the ab-
sence of exons B7 and B10 in Kelly (Fig. 4b), how-
ever, indicates that either the deletions are more
complex than what was originally predicted by the
array CGH data (Stallings et al., 2006) or that a
combination of deletion and aberrant splicing has
led to the noncontiguous expression of these
exons. Apart from Kelly and SK-N-AS, there was
no evidence for homozygous deletion of PTPRD in
the other cell lines or tumors using either array
CGH or real time PCR analyses. One cell line, SK-
N-DZ had a hemizygous deletion of a large seg-
ment of the 9p which includes PTPRD, as ascer-
tained by CGH (Schleiermacher et al., 2003). This
cell line is missing exons B2, B4, B5, B7, B8, and
B11 of the 50 UTR in PTPRD cDNA (Fig. 4d). To
Figure 3. Analysis of exons from the 50 region of PTPRD in cDNAderived from brain (A) and kidney (B). Forward primers from exons B2,B4, B5, B7, B8, B10, and B11 from the 50 UTR or coding sequencesexons E2 and E3 were used in conjunction with a reverse primer fromexon 5. Primers are listed in the supplemental information section. Asexpected, each forward primer from a 50 UTR exon produced a PCRproduct, decreasing in size from exons B2 through B11 from braincDNA. Forward primers from coding exons E2 and E3 also producedPCR products of decreasing size. No PCR products were observedwith 50 UTR forward primers from kidney, which has been reported topossess only the S isoform of PTPRD (lacking the 711 bp 50 UTR). PCRproducts were observed, however, using forward primers from codingexons E2 and E3.
Figure 4. Analysis of PTPRD 5’ exons in cDNA derived from theneuroblastoma cell lines CHP-212 (A), Kelly (B), SK-N-BE (C), and SK-N-DZ (D) using forward primers from exons B2, B4, B5, B7, B8, B10,and B11 from the 50 UTR or coding sequences exons E2 and E3 in con-junction with a reverse primer from exon E5, as described in Figure 2.CHP-212 (A) had a splicing pattern similar to that of normal brain tis-sue, with all primer sets producing PCR product. Kelly cells lackedexons B7 and B10, while retaining exon B8. The absence of B7 and B10is due to complex homozygous deletion of corresponding regions ofgenomic DNA. Both SK-N-BE and SK-N-DZ lacked exons B2, B4, B5,B7, B8, and B11, presumably through aberrant splicing of mRNA. Inaddition, SK-N-DZ had an exon 3 fragment that was larger then theexpected size, perhaps representing either additional mRNA splicingaberrations or a PCR generated artifact. Neither cell line had homozy-gous deletion of any part of the PTPRD gene (Fig. 4).
Genes, Chromosomes & Cancer DOI 10.1002/gcc
200 NAIR ET AL.
determine whether small-scale cryptic deletions of
the PTPRD region might exist on the ‘‘non-
deleted’’ chromosome 9 homologue, which might
have escaped detection by CGH analysis, we per-
formed real time PCR analysis on genomic DNA
using 13 primers, including primers from exons B2,
B3, B4, B5, B6, B7, and B8 (Fig. 5). Although the
chromosome 9 hemizygous deletion was apparent
from the PCR analysis, there was no evidence for
homozygous deletion, indicating that some form of
aberrant mRNA splicing led to the loss of the these
50 UTR exons in cDNA. The SK-N-BE cell line,
which had a similar expression profile for the 50
UTR exons as SK-N-DZ, was also analyzed for
possible deletion of small regions of the PTPRDgene (Fig. 5). No evidence for either hemi- or
homozygous deletion was obtained, confirming
that aberrant splicing was responsible for the miss-
ing exons in the cDNA.
DISCUSSION
The loss of 50 UTR exons in PTPRD mRNA
occurs at high frequency (>50%) in NBL tumors
and cell lines as a consequence of either homozy-
gous genomic deletion or aberrant mRNA splicing.
The high frequency of exon loss strongly impli-
cates this gene, and particularly the 50 region of
this gene, in NBL pathogenesis. Both microarray
expression and real time PCR analyses of exon
regions that are not deleted or aberrantly spliced
indicate that the expression of this gene has been
decreased in NBL from patients with high stage
disease. The possibility that loss of these 50 UTR
exons decreases mRNA stability is consistent with
these results and warrants further study. It is possi-
ble that multiple mechanisms might exist for down
regulating PTPRD activity in high stage NBLs,
given that tumors and cell lines that express the
entire set of 50 UTR exons also have relatively low
levels of PTPRD mRNA. Aberrant hypermethyl-
ation of the promoter region of this gene might be
another mechanism for down-regulation.
Several lines of evidence indicate that PTPRDmight be acting as a metastasis suppressor gene, as
opposed to an initial suppressor of tumorigenesis
in NBL. First, we have demonstrated that in a
matched primary and metastatic tumor sample
only the metastatic sample had microdeletion of
this gene (Stallings et al., 2006). Secondly, the fact
that primary tumors derived from patients with
metastatic disease have lower expression of
PTPRD relative to loco-regional tumors further
suggests that inactivation of this gene is important
for metastasis. It is also noteworthy that PTPRD
interacts with a putative metastasis suppressor,
MIM, which is involved with cytoskeletal remodel-
ing (Woodings et al., 2003; Gonzalez-Quevedo
et al., 2005).
The involvement of PTPRD in several forms of
cancer, as indicated by both microdeletions and
mutations (Sato et al., 2005; Zhao et al., 2005; Sjo-
blom et al., 2006; Stallings et al., 2006; Purdie
et al., 2007; Stark and Hayward, 2007) should initiate
Figure 5. Real time quantitative PCR analysis of genomic DNA using13 primer sets (see supplemental section on primers) spanning thePTPRD gene. MJ90 is a diploid human fibroblast cell line used as a con-trol, while SK-BN-BE and SK-N-DZ are neuroblastoma cell lines. Ahemizygous deletion was evident for all primer sets in SK-N-DZ (whichhas been reported to be hemizygously deleted for a large segment of
chromosome 9p by CGH (Schleiermacher et al., 2003), while no dele-tion for PTPRD was detected in SK-N-BE. The absence of a homozygousdeletion in these cell lines indicates that the missing 50 UTR exons fromthe PTPRD cDNA (Fig. 4) resulted from mRNA splicing. GAPDH wasused for normalization. [Color figure can be viewed in the online issue,which is available at www.interscience.wiley.com.]
Genes, Chromosomes & Cancer DOI 10.1002/gcc
201ABERRANT SPLICING OF PTPRD IN NEUROBLASTOMA
a greater interest for this gene in future studies.
The role of the 50 UTR should be a particular pri-
ority given that some tumors have microdeletions
affecting only this region or have splice variants
that basically mimic these deletions.
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