Am. J. Hum. Genet. 41:202-217, 1987
Genetic Mechanisms of Tumor-Specific Loss of 1 'p DNA
Sequences in Wilms Tumor
DAT D. DAO,* WANDA T. SCHROEDER,* LIAN-YU CHAO,* HIDEAKI KIKUCHI,*
LOUISE C. STRONG,t VINCENT M. RICCARDI,§ SEN PATHAK,4
WARREN W. NICHOLS," WILLIAM H. LEWIS,
AND GRADY F. SAUNDERS*
Departments of *Biochemistry and Molecular Biology, tPediatrics, and *Genetics, The
University of Texas M. D. Anderson Hospital and Tumor Institute; and §Research Cytogenetics
Laboratory, Baylor College of Medicine, Houston, TX; IlDepartment of Safety Assessment,
Merck, Sharp and Dohme Research Laboratory, West Point, PA; and #Department of Surgery,
University of Toronto, Toronto, Canada
Wilms tumor, a common childhood renal tumor, occurs in both a
heritable and a nonheritable form. The heritable form may occasion-
ally be attributed to a chromosome deletion at Ip13, and tumors from
patients with normal constitutional chromosomes often show deletion
or rearrangement of 1lpl3. It has been suggested that a germinal or
somatic mutation may occur on one chromosome 11 and predispose to
Wilms tumor and that a subsequent somatic genetic event on the
normal homologue at 1 Ip13 may permit tumor development. To study
the frequency and mechanism of such tumor-specific genetic events,
we have examined the karyotype and chromosome 11 genotype of
normal and tumor tissues from 13 childhood renal tumor patients with
different histologic tumor types and associated clinical conditions.
Tumors of eight of the 12 Wilms tumor patients, including all viable
tumors examined directly, show molecular evidence of loss of lip
DNA sequences by somatic recombination (four cases), chromosome
loss (two cases), and recombination (two cases) or chromosome loss
and duplication. One malignant rhabdoid tumor in a patient heterozy-
gous for multiple lip markers did not show any tumor-specific lip
alteration. These findings confirm the critical role of lip sequences in
Received November 11, 1986; revised January 26, 1987.
Address for correspondence and reprints: Dr. Grady F. Saunders, Department of Biochemistry
and Molecular Biology, The University of Texas System Cancer Center, 6723 Bertner Avenue,
Houston, TX 77030.
© 1987 by the American Society of Human Genetics. Allrightsreserved. 0002-9297/87/4102-0011$02.00
Loss OF 11P DNA SEQUENCES IN WILMs TUMOR
Wilms tumor development and reveal that mitotic recombination may
be the most frequent mechanism by which tumors develop.
Wilms tumor is among the more common childhood solid tumors, occurring at
a rather stable rate of 1/10,000 live-born children in most areas reported (Mat-
sunaga 1981). While most tumors are sporadic and occur in children with nor-
mal karyotypes and no constitutional anomalies, rare familial cases and cases
associated with aniridia and a constitutional chromosome lp deletion suggest
that some fraction of Wilms tumors may be attributable to heritable predispos-
ing factors (Knudson and Strong 1972; Riccardi et al. 1978). A two-hit mutation
model was proposed to account for hereditary and nonhereditary forms of
Wilms tumor, suggesting that an initial genetic event might occur in a germinal
or somatic-cell line. In either case a subsequent somatic event would be neces-
sary for tumor development and might involve mutation at the homologous
gene locus (Knudson and Strong 1972). The occurrence of Wilms tumor in
50%-60% of patients with aniridia and chromosome Ip13 deletion suggests a
possible chromosomal site for genes critical to Wilms tumor development (Tur-
leau et al. 1984). The notion that a tumor-specific somatic genetic event might
also involve chromosome lip has recently been supported by the demonstra-
tion oftumor-specific loss ofheterozygosity for lip loci even in the presence of
apparently normal chromosomes 11 (Fearon et al. 1984; Koufos et al. 1984;
Orkin et al. 1984; Reeve et al. 1984). We have investigated the frequency ofthis
loss-and the mechanism by which the loss of lp DNA sequences occurs-by
using a series of gene probes that mapped to chromosome 11 (HGM 8 1985),
e.g., human oncogene c-Harvey ras-1 (HRAS1;
11p15.5), parathyroid hormone (PTH; llpl5.4-pl5.l), catalase (CAT; I1p13),
pepsinogen (PGA; lpi I-q13), and apolipoprotein Al (APOAl; 1 1q13). These
probes were used to examine tumor-specific changes in the DNA of 13 child-
hood renal tumor patients. Multiple mechanisms by which tumors lose loci in a
series of Wilms tumor patients are demonstrated in this study.
lIpl5.5), insulin (INS;
MATERIAL AND METHODS
Tumor and normal tissue samples were collected from 13 patients with renal
tumors. The clinical characteristics, histologic classifications, and specimen
types are listed in table 1. Twelve tumors were typical Wilms tumors, having
blastemal, epithelial, and stromal elements and no anaplasia; one tumor was a
malignant rhabdoid tumor.
DNA was isolated from primary untreated tumor specimens in six cases,
from a posttreatment necrotic tumor specimen in one case, from primary and
metastatic tumor specimens in two cases, and from tissue culture in four cases.
Constitutional DNA was obtained from peripheral blood lymphocytes trans-
formed by Epstein-Barr virus, from primary skin fibroblast cultures, and from
.. Q. +41,
Z Z.w W
Loss OF 1 1P DNA SEQUENCES IN WILMS TUMOR
normal kidney or other surgical specimens. When possible, normal tissue and
tumor karyotypes were obtained.
Peripheral lymphocytes obtained from Wilms tumor patients were grown in
RPMI-1640 medium supplemented with 15% fetal calf serum and phytohemag-
glutinin (PHA) at 37 C for 72 h and were harvested following the standard air-
drying technique (Moorhead et al. 1960). Tumor biopsies were processed in
either of the following two ways: (1) the specimen was first dissociated with
collagenase (collagenase N; 0.75 mg/ml) and single-cell suspensions were then
set up for direct harvest or short-term culture (24-48 h); or (2) the cell frag-
ments were explanted as described elsewhere (Riccardi and Elder 1986), and
the tumor-harvesting procedure was either the same as that used for the lym-
phocyte culture or in situ on tissue-culture slides. Various banding patterns
were induced by following the routine techniques described elsewhere (Pathak
Seven recombinant plasmid clones containing human sequences of HRAS1,
INS, PTH, FSHB, CAT, PGA, and APOAl, all previously mapped to chromo-
some 11, were used in this study.
The pEJ probe contained a 6.6-kb BamHI fragment of HRAS1 oncogene
subcloned into pBR322 (Shih and Weinberg 1982). The phINS 300 gene probe
contained a 5-kb XhoI fragment including the INS gene and 2 kb of 5'- and 1.5
kb of 3'-flanking nucleotides inserted in the Sail site of pBR322 (Bell et al.
1981). The pPTHm122 gene probe was a cloned cDNA encoding human prepro-
parathyroid hormone (Hendy et al. 1981). The CAT gene probe was either a
1.1-kb BamHI/HindIII fragment obtained from the pCAT41 cDNA recombi-
nant plasmid (Korneluk et al. 1984) or an 800-bp ScaI/SnaBI intron fragment
subcloned in pSP64 (Quan et al. 1985). The APOA1 gene probe was a 2.2-kb
PstI genomic fragment cloned into pUC8 vector (Kessling et al. 1985). APOAl
has been mapped to 1 1q13 (Schroeder and Saunders, in press). The PGA gene
probe (Taggart et al. 1985) contained a 705-bp cDNA insert containing 686 bp of
sequence corresponding to a portion of exon 3-exon 8 of a nine-exon human
pepsinogen gene. This probe hybridizes to exons 3-8 of the PGA5, PGA4, and
PGA3 genes. The pFSH-1.4 probe was a 1.4-kb PstI fragment containing the
structural gene for 3-follicle-stimulating hormone (FSHB) (Glaser et al. 1986).
The anonymous polymorphic DNA probe DIS1, originally called XH3, was
isolated from a human genomic library in bacteriophage XCharon 4A by Harper
and Saunders (1981).
DNA samples from Wilms tumor patients were isolated from peripheral
blood leukocytes; lymphoblastoid cell lines from leukocytes transformed by
Epstein-Barr virus; surgical or autopsy specimens of skin, primary tumors,
metastatic tumors, and other normal tissues; and long-term cell cultures from
tumor or skin fibroblasts. Leukocytes from blood, 4,000 g pellets of kidney
homogenate, and cell pellets of cell cultures were subjected to DNA isolation
as described elsewhere (Gray et al. 1985), with the following modifications. In
brief, the pellets were suspended in SSC (0.15 M sodium chloride, 0.015 M
sodium citrate) + 5 mM ethylenediaminetetraacetate (EDTA) + 0.2% sodium
dodecyl sulfate (SDS) and then treated with proteinase K (200 pug/ml) at 50 C
for >4 h. The DNA was extracted with phenol saturated with TE buffer and
Sevag (chloroform:isoamyl alcohol; 24:1) and precipitated with ethanol.
Southern Blotting and Labeling ofDNA
Restriction digestions ofDNA samples from Wilms tumor and normal tissues
were performed with Avall, BamHI, BglII, EcoRI, HaeIII, HindII1 + PstI,
KpnI, PstI, SstI, TaqI, and XmnI at 2-4 U enzyme/pg DNA. The digested
DNAs were then electrophoresed through agarose gels and transferred by blot-
ting to nitrocellulose membranes as described elsewhere (Southern 1975). The
immobilized DNAs were prehybridized with 6 x SSC, 5 x Denhardt's solution
(0.1% bovine serum albumin, 0.1% Ficoll, 0.1% polyvinylpyrrolidone), 100 pug
heat-denatured sonicated salmon sperm DNA/ml, and 0.5% SDS at 68 C for 4-
6 h. Hybridization was carried out using the same solution containing human
gene probes labeled either by in vitro nick-translation with [a-32P]dCTP (Lai et
al. 1979) or by an oligolabeling technique (Feinberg and Vogelstein 1983) to
specific activities >108cpm/tLg.The filters were then washed with several
changes of SSC, 0.1% SDS, and 0.2 x SSC, 0.1% SDS at 68 C and exposed to
X-ray film at -70 C for several days. Alternatively, 50% formamide was used
in the prehybridization and hybridization solutions at 42 C. Similar results were
obtained with either hybridization procedure.
Copy Number ofChromosome 11 Gene Markers
To determine the relative gene copy number at a given locus in tumor and
normal tissue, quantitative scanning densitometric analysis was applied to the
hybridization intensity obtained with the PTH gene probe and a probe homolo-
gous to the HLA class II-associated invariant-chain gene (7-2D). The HLA
class Il-associated invariant-chain gene has been mapped to chromosome 5.
This gene was selected because (1) no restriction-fragment-length polymor-
phism (RFLP) has been observed in samples for >40 individuals (Kudo et al.
1985); (2) no chromosome 5 abnormalities have been observed in Wilms tumors
in the present series or others (Slater 1986); and (3) the EcoRI digest gives only
one hybridization band each-a 3.7-kb band with the PTH gene probe and an
8.3-kb band with the 7-2D probe. For each patient, DNAs from normal tissue
and from Wilms tumor were digested to completion with EcoRI and frac-
tionated on an agarose gel. After transfer to nitrocellulose membranes, the
immobilized DNAs were hybridized simultaneously with probes for PTH and
for the HLA class Il-associated invariant-chain gene. The hybridizing bands
were scanned with a densitometer, and the hybridization intensities of PTH
restriction fragments in the normal and the tumor DNAs were normalized with
the hybridization intensities of the 7-2D bands.
DAO ET AL.
Loss OF 11P DNA SEQUENCES IN WILMs TUMOR
Normal and tumor tissues from 13 childhood renal tumor patients were ex-
amined for karyotype, DNA sequences from lIp and 1 lq, and gene copy num-
ber. All 13 patients were heterozygous in their constitutional tissues for at least
one lp gene. Eight tumors, including all the viable Wilms tumors that were
examined directly, showed loss of heterozygosity for lip markers. These
findings indicated that the loss of I lp sequences could be attributed to several
Tumor-specific Loss of lip Heterozygosity via Chromosome Loss
Patient 1 had a normal constitutional karyotype, but her tumor karyotype
revealed abnormalities both on direct examination and in short-term culture.
These included a duplication of lq21-qter and deletion of lp33-pter, as well as
loss of one chromosome 11 and the presence of a ring chromosome and an
acentric fragment (table 1). The hybridization patterns of PTH with this pa-
tient's DNA showed that her normal cells were heterozygous for PTH in
PstI+Hindll-digested DNA with 2.7-kb and 2.1-kb fragments. Her tumor
DNA revealed only the 2.1-kb fragment (fig. 1A, table 2). To determine
Loss of Heterozygosity by Chromosome Loss
in a Wilms' Tumor
PTH/Pst I + Ll
FIG. 1.-Loss of heterozygosity by chromosome loss in Wilms tumor. A, Hybridization pattern
of [32P]-labeled PTH cDNA to DNAs from patient 1. Ten micrograms each of normal kidney (NK)
and Wilms tumor (WT) DNAs were restriction digested with PstI + HindIII, subjected to elec-
trophoresis on a 1.0% agarose gel, and transferred to nitrocellulose membrane. Hybridization with
[32P]-labeled PTH cDNA (specific activity 108 cpm/Lg) was carried out at 68 C as described in the
Material and Methods section. The filter was washed and dried, and bands were visualized by
means of autoradiography. B, Densitometric scanning of Southern hybridization with lIp probe
(PTH) and with chromosome 5 HLA class II-associated invariant-chain gene (7-2D). Ten mi-
crograms each of NK and WT DNAs were restriction digested with EcoRI and subjected to
Southern blotting as described above. Hybridization with [3IP]-labeled PTH cDNA and [32p]_
labeled 7-2D was carried out as described (Southern blot not shown). Densitometric scanning was
normalized with hybridization intensities of7-2D as described in the Material and Methods section.
The thinner line on the graph (
) denotes the reading for normal tissue; the thicker line
) denotes the reading for tumor tissue.
In * m u
iQ oR ~~zz
e 3 e3
m oo . oo oooooooeno0
;0 E : kb :: ::~~~~~9O
s o O5 O
00 00 00 00
.. .- Z
whether the tumor was hemizygous for the PTH locus-and presumably for
other chromosome 11 sequences, as suggested by the tumor karyotype-the
relative gene copy number was examined. The extent of hybridization of
the HLA class II-associated invariant-chain gene from chromosome 5 was the
same in normal and tumor samples, while the intensity of the PTH hybridiza-
tion in tumor was reduced to approximately one-half that in normal kidney (fig.
1B). The tumor karyotype, PTH genotype, and gene dosage support the net
loss of chromosome lip DNA sequences.
Tumor-specific Loss of lip Heterozygosity via Somatic Recombination
Patient 2 had a normal constitutional karyotype (table 1) and was heterozy-
gous at the loci defined by HRAS1, PTH, CAT, and APOAl (fig. 2, table 2). No
metaphases were recovered from the tumor or subsequent vaginal metastasis.
However, analyses of restriction-enzyme digests of DNAs from the primary
tumor and from metastases obtained at two different times revealed loss of the
6.6-kb allele for HRAS1, loss of the 2.7-kb allele for PTH, and loss of the 12.0-
kb allele for CAT but retention ofboth the 6.6-kb and 8.3-kb alleles for APOAl
(fig. 2A-D, table 2). No change in the hybridization intensity of PTH and the
HLA class II-associated invariant-chain gene in tumor and normal tissue was
observed, suggesting that the tumor had two copies of the PTH gene (fig. 2E).
Loss of heterozygosity for I lp markers in the presence of normal gene copy
number, with maintenance of heterozygosity for an
somatic recombination on chromosome 11 between the CAT and APOAl re-
gions. Tumor and metastases resected at different times showed the same
genotypic pattern, consistent with clonal development of the tumor and metas-
To determine whether the loss of heterozygosity in patient 2 was due to a
tumor-specific loss of lip or to a loss of random chromosome sequences, a
polymorphic marker on chromosome 3, DIS1 (Harper and Saunders 1981;
Goode et al. 1986), was used to examine DNAs from this patient. The hybridi-
zation patterns of DISI with HindIII-digested DNAs from patient 2 demon-
strated retention of heterozygosity in tumor tissue (data not shown).
After restriction-enzyme digestion with appropriate enzymes as shown in
table 2, tumor DNA from patient 3 revealed loss of the 2.1-kb PTH allele and
the 2.5- and 1.0-kb CAT allele but retention of both APOAl alleles (table 2).
Tumor-DNA digests from patient 4 revealed loss of the 13.8-kb INS band, and
the CAT bands 1.9 kb and 3.5 kb in Avall digest; however, the bands repre-
senting the PGA B and C phenotypes were present (table 2). Comparable
tumor-DNA digests from patient 5 showed loss ofthe 13.8-kb INS allele but the
presence of both CAT alleles (table 2). Vis-a-vis the HLA class II-associated
invariant-chain gene, no change in hybridization intensity between normal and
tumor tissue from these patients was observed for PTH. The tumor-specific
loss of heterozygosity without reduction in gene copy number for the lip
marker, with retention of heterozygosity for the more proximal lip or 1lq
marker, suggested chromosome II recombination.
lIq marker, suggested
DAO ET AL.
Loss OF 11P DNA SEQUENCES IN WILMS TUMOR
Loss of Heterozygosity by Somatic Recombination in a Wilms' Tumor
z 5 1
PTHiPst. I + HindIII
Apo Al / Xmn I
FIG. 2.-Loss of heterozygosity, by somatic recombination, in a Wilms tumor. Hybridization
pattern on (A) [32P]-labeled HRASI gene, (B) PTH cDNA, (C) CAT cDNA, and (D) APOAl
genomic fragment to DNAs from patient 2. Ten micrograms each of normal kidney (NK), Wilms-
tumor (WT), and vaginal metastasis (Vag. Met.) DNA were restriction digested with (A) BamHI,
(B) PstI + HindIII, (C) KpnI, and (D)XmnI and electrophoresed on 1% agarose gels. Details are as
given in fig. 1. Hybridization was carried out as described in the Material and Methods section. A
faint hybridization band in the vaginal metastasis sample could likely represent contamination with
normal tissue. E, Densitometric scanning ofSouthern hybridization with IIp probe (PTH) and with
chromosome 5 HLA class II-associated invariant-chain gene (7-2D). Details are as given in fig. 1.
The thinner line on the graph (
(-) denotes the reading for tumor tissue.
) denotes the reading for normal tissue; the thicker line
Tumor-specific Loss ofIlp Heterozygosity with Retention ofNormal
Gene Copy Number: Chromosome Loss and Duplication
or Somatic Recombination
Hybridization of tumor DNA from patient 6 with the PTH and APOAI gene
probes after appropriate restriction-enzyme digestion revealed tumor-specific
loss of the 2.1-kb allele for PTH and of the 8.3-kb allele for APOAl (table 2).
EcoRI- and TaqI-digested DNA from patient 7 revealed tumor-specific loss of
heterozygosity for the INS gene and for the CAT intron sequence (table 2).
Hybridization intensity for PTH and the HLA class II-associated invariant-
chain gene were similar in normal and tumor tissue from these two patients
(data not shown). These findings confirm tumor-specific lip alteration but do
not distinquish between somatic recombination and chromosome loss with
Loss of lip DNA Sequences via Chromosome Loss in One of Two Tumors
from the Same Patient
Two tumors were removed from the right kidney of patient 8 during surgery.
The left kidney had been resected previously for Wilms tumor. The first tumor
(TI)was from the upper pole whereas the second tumor (T2) was from the lower
pole (table 1). These two tumors could be clonal in origin or result from two
primary events. Hybridization of T1 DNA displayed retention of heterozygos-
ity for polymorphic markers on chromosome 11. On the other hand, hybridiza-
tion of T2 DNA from this patient with the CAT and APOA1 gene probes after
appropriate restriction-enzyme digestions revealed tumor-specific loss of the
1.9-kb allele for CAT and of the 6.6-kb allele for APOAl (table 2). Gene-dosage
studies with a probe for FSHB, located in llpl3, and with the 7-2D standard
demonstrated that, vis-a-vis normal tissue, there was only one copy ofthe FSH
allele-and presumably other chromosome 11 alleles-present in the tumor
tissue. Since the region from FSH to APOAl spans the centromere, chromo-
some deletion in this case is unlikely. Therefore, we concluded that this was
due to loss of a chromosome 11 in tumor tissue.
Loss of lip DNA Sequences via Constitutional Chromosome 11 Deletion
Patient 9 had aniridia, a de novo constitutional deletion of chromosome 11
(p1 .2p 14. 1), and one-half the normal level of red blood cell catalase activity
(R. E. Ferrell, personal communication). His normal cells showed heterozy-
gosity for HRAS1 and APOAl (data not shown). The only available tumor cells
were from a long-term culture that had a karyotype identical to that of his other
tissues. No tumor-specific loss ofDNA sequences was observed in this culture.
Tumors with No Apparent Loss of lip DNA Sequences
Patients 10-12 were studied in a similar manner. Normal tissues from each
individual demonstrated a normal karyotype (table 1) and heterozygosity for at
least one lip gene marker. The available tumor tissue was not optimal, includ-
ing late passage, somewhat fibroblastic cultures (patients 10 and 11), and exten-
sively necrotic Wilms tumor resected postchemotherapy with no viable tumor
cells on histologic examination (patient 12). DNA from the long-term cultures
and from the necrotic Wilms tumor did not reveal any change in the presence of
chromosome 11 DNA sequences.
Multiple tissue samples were obtained from autopsy from a patient with a
malignant rhabdoid tumor (patient 13). The patient had a normal constitutional
karyotype (table 1) and was heterozygous for multiple chromosome 11 genetic
loci, including HRAS1, INS, PTH, CAT, and APOAl (data not shown). Exami-
nation ofDNA from the primary rhabdoid tumor and from lung metastases did
not reveal alteration at any of these loci.
DAO ET AL.
Loss OF 1lP DNA SEQUENCES IN WILMS TUMOR
By means of cytogenetic and molecular analyses using a series of markers
from different regions of chromosome 11, the present study has examined the
frequency, specificity, and mechanism of loss of lip DNA sequences in Wilms
tumor. The patient series included 12 individuals representative (by age, sex,
and tumor histology; Breslow and Beckwith 1982) of Wilms tumor patients in
general and one individual with malignant rhabdoid tumor.
Four of the Wilms tumor patients had a probable germinal mutation or pre-
disposition to Wilms tumor. Patient 9, who had a constitutional chromosome 11
deletion (p1 1.2-p14. 1), did not show tumor-specific loss ofDNA sequences in
tumor cells from a long-term culture. Any somatic event, detectable by our
methods, involving loss of the wild-type allele from the normal homologue
would render the cell devoid of genetic material for a large region, a condition
that seems unlikely to be viable. Hence, it is not surprising that, with the
techniques available to us, the tumor did not show alteration in the ostensibly
normal lIp. Similarly, the tumor karyotype from another Wilms tumor/aniridia
patient with a constitutional deletion 11 (pl2pl4) revealed no additional abnor-
mality of chromosome 11 (Nakagome et al. 1984).
Patient 8 had Perlman syndrome, a rare autosomal recessive condition asso-
ciated with nephroblastomatosis and Wilms tumor (Greenberg et al. 1986). The
loss of chromosome 11 in one tumor confirms the role of lIp sequences in
Wilms tumor even in this rare, autosomal recessive syndrome; the genetic
differences in T1 and T2 confirm that each tumor developed independently, the
results of different genetic events.
Patient 3 had bilateral Wilms tumor, Drash syndrome, and a gonadoblas-
toma; and patient 4 had a fetal rhabdomyomatous Wilms tumor-a rare type
that has a somewhat distinct young age of onset, a high frequency of bilat-
erality, and occasionally has been associated with aniridia (Wigger 1976; Gon-
zales-Crussi et al. 1981)-and multiple genitourinary anomalies. Tumor-
specific loss of heteozygosity for markers from chromosome lp occurred in
both cases and could be attributed to somatic recombination, occurring be-
tween CAT and APOAI in patient 3 and between CAT and PGA in patient 4.
Eight patients had sporadic unilateral Wilms tumor in the absence of con-
genital anomalies. Five ofthe eight sporadic unilateral Wilms tumors, including
all viable tumors examined directly, showed loss of lp DNA sequences, pre-
sumably loss ofa wild-type allele following a somatic 1ip mutation. Tumor and
metastases resected at different times from patient 2 showed the same geno-
typic pattern, consistent with clonal development ofthe tumor and metastases.
The common finding ofloss ofthe lp DNA sequences in tumors from patients
with different associated anomalies-and perhaps different predisposing ge-
netic factors-suggest a common critical genetic pathway to Wilms tumor.
Examination of tumor DNA from patient 2 with a gene probe from chromo-
some 3 demonstrated retention of heterozygosity in Wilms tumor tissue. Al-
though we did not examine DNA markers for other chromosomes, tumor-
specific loss of heterozygosity for markers on chromosome 3 (in the present
study) and on chromosomes 1, 13, 14, 15, 17, or 20 (in other studies: Orkin et al.
1984; Reeve et al. 1984) has also not been observed. From the results with
the tumor karyotypes in this series, we would anticipate a loss of heterozy-
gosity for markers on chromosome 1 (p33-pter) in patient 1, on chromosome 1
(qter-pl2) in patient 5, and on chromosome 16 (pll.2-qter) in patient 5. Al-
though these regions appear to show nonrandom loss in Wilms tumor (Slater
1986), these findings are not as consistent as the loss of lp DNA sequences.
The only viable tumor examined directly that did not show loss of lp DNA
sequences was a malignant rhabdoid tumor, a rare childhood renal tumor with
distinct clinical, histologic, and demographic characteristics (Palmer and
Sutow 1983; Vogel et al. 1984). There was adequate opportunity to detect
alteration in I lp sequences, because patient 13 was heterozygous for multiple
I lp markers. Ifthe absence ofevidence of lIp events in rhabdoid tumor can be
confirmed in other cases, it may indicate that this tumor is different from Wilms
tumor genetically as well as clinically.
A model analogous to retinoblastoma (Strong et al. 1981; Cavenee et al. 1983,
1985; Dryja et al. 1984) has been developed for Wilms tumor, a model in which
a constitutional chromosomal 1 1p13 deletion predisposes to Wilms tumor with
a 50%-60% probability of tumor and a high frequency of bilateral tumors
(Knudson and Strong 1972; Turleau et al. 1984). A tumor-specific somatic event
involves either a cytogenetically visible lIp13 deletion or rearrangement in
-30% of cases (Slater 1986) or loss of 1 lp DNA sequences demonstrated by
loss of heterozygosity for lIp15 markers in some 50% of cases (Koufos 1984;
Orkin et al. 1984). In previous work we obtained evidence suggesting that the
loss of maternal alleles in Wilms tumor is not a random event (Schroeder et al.
1987). In the present series, which used markers from different regions of
chromosome 11, evidence supports the hypothesis that I lp alteration occurs in
most Wilms tumors. No single genetic locus was either sufficiently poly-
morphic or so consistently involved as to demonstrate lIp alteration in every
tumor. Multiple marker studies revealed that the most frequent mechanism for
tumor-specific loss of heterozygosity with apparent new germinal or somatic
initial mutations was somatic recombination. Recombination was observed or
could be inferred at different regions between the presumptive Wilms tumor
locus and CAT, between CAT and PGA, or between CAT and APOAI, so no
specific hot spot for recombination was detected. If we assume that the critical
region involved is the putative Wilms tumor gene in IIp13, then these data are
consistent with the mapping of the Wilms tumor locus distal to CAT (Van
Heyningen et al. 1985; Glaser et al. 1986).
We thank Drs. R. A. Weinberg, G. Bell, H. Kronenberg, R. A. Gravel, and R. T.
Taggart for the generous gifts of plasmids pEJ, phINS300, pPTHml22, CAT intron
gene, and phpepl4-21, respectively. We also thank Integrated Genetics for the generous
gifts ofpFSH-1.4, Drs. S. Humphries and A. Kesslingfor the pApAl gene probe, Dr. H.
Evans for review of the histology of the Wilms tumors, and Dr. R. Ehrlich for tissue
samples on patient 3. Thanks also to D. W. Elder and Dr. D. H. Lockwood for assis-
tance with cell cultures and cytogenetic preparation. This work was supported by the
DAO ET AL.
Loss OF 11P DNA SEQUENCES IN WILMS TUMOR
National Institutes of Health (CA 34936 and CA 33624), the American Cancer Society
(ACS-CD 223), and the Robert A. Welch Foundation.
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