We examined 18 atypical teratoid and rhabdoid tumors of the brain and 7 renal and 4 extrarenal rhabdoid tumors for mutations in the candidate rhabdoid tumor suppressor gene, INI1. Fifteen tumors had homozygous deletions of one or more exons of the INI1 gene, and the other 14 tumors demonstrated mutations. Germ-line mutations of INI1 were identified in four children, one with an atypical teratoid tumor of the brain and three with renal rhabdoid tumors. These studies suggest that INI1 is a tumor suppressor gene involved in rhabdoid tumors of the brain, kidney, and other extrarenal sites.

Rhabdoid tumor was originally described as a “rhabdomyosarcomatous variant” of Wilms’ tumor in the kidney (1), although it soon became apparent that the morphological, immunohistochemical, and biological features of rhabdoid and Wilms’ tumors were distinct (2, 3). The occurrence of rhabdoid tumors in sites such as the brain, skin, liver, thymus, and orbit were later reported, and considerable debate ensued as to whether renal and extrarenal rhabdoid tumors were a heterogeneous group of neoplasms with some common cytological features or a distinct tumor type with a unique origin (4). Rhabdoid tumors in all sites tend to present at an early age, generally between birth and 2 years, and have an extremely aggressive course.

The most common location for extrarenal rhabdoid tumor is the CNS3(4, 5). In the brain, the tumors may be composed purely of rhabdoid cells, or they may present as a mixture of rhabdoid tumor with PNET, mesenchymal, and/or epithelial elements, an entity referred to as ATT (5). Because a large proportion of the CNS ATTs contain fields resembling PNET, pathologists have routinely misclassified AT/RTs of the CNS as MB or PNET.

Cytogenetic studies of rhabdoid tumors provided the first evidence that there was a common genetic basis for CNS, renal, and extrarenal rhabdoid tumors. We reported monosomy or deletion of chromosome 22 as the primary cytogenetic change in AT/RT of the brain (6) and, based on the identification of overlapping deletions of a region within 22q11, proposed that loss or inactivation of a tumor suppressor gene in chromosome 22 was also responsible for the initiation or progression of rhabdoid tumors in other sites (7). Recently, Versteege et al.(8) reported deletions and somatic mutations in the hSNF5/INI1 gene, which maps to 22q11.2, in 12 of 13 renal or extrarenal rhabdoid tumors. Although germ-line mutations in INI1 were not observed, the homozygous loss or inactivation of the INI1 gene is consistent with its function as a tumor suppressor gene.

The purpose of this study was to determine whether INI1 is a candidate tumor suppressor gene for AT/RTs of the brain. The presence of inactivating mutations in INI1 would support our hypothesis that there is a similar genetic etiology for rhabdoid tumors of all sites. The demonstration of germ-line mutations in patients with rhabdoid tumors from the brain or kidney would lend additional support to this hypothesis and would allow for genetic testing in families at risk for developing these tumors.

Tumor Specimens.

Biopsies were obtained from patients at The Children’s Hospital of Philadelphia or other hospitals, as well as from the Cooperative Human Tissue Network and National Wilms’ Tumor Study Group tumor banks. Cases 86-18 and 88-128 (6), 97-55,97-64 and 96-10-P308 (9), and 97-101 (10) and 97-102 (11) have been studied previously. Histological diagnoses were confirmed for all outside CNS cases by L. B. R. The SJSC rhabdoid tumor cell line has been studied previously (7).

DNA and RNA Isolation.

Human genomic DNA was isolated from lymphocytes using an automated DNA extractor (341 Nucleic Acid Purification System; Applied Biosystems). Cosmid DNA was isolated from overnight cultures by a standard alkaline lysis method. Total RNA was isolated from tumor tissue or tumor cell lines using TRIzol (Life Technologies, Inc.), and DNA from normal and tumor tissue was extracted with DNAzol (Life Technologies, Inc.).

Molecular Cytogenetic Studies.

Chromosomes were harvested from biopsy specimens, peripheral blood lymphocytes, or lymphoblastoid cell lines according to established procedures. DNA probes were isolated from the LLNCO3 library or were obtained from Oncor (Gaithersburg, MD). FISH was performed as described previously (7). The slides were examined by two investigators on a Zeiss axioplan microscope with dual- or triple-band pass filter sets, and images were collected with Smartcapture software (Vysis, Inc).

Isolation of Cosmid Clones for the INI1 Gene.

Filters from the chromosome 22-only cosmid library LLNCO3 were screened by hybridization with P-32 labeled PCR products from exons 1, 4, and 9 from the INI1 gene. Each positive clone was tested by PCR to determine which INI1 exons were present.

Microsatellite Analysis.

Paired tumor DNA and lymphoblast DNA samples were analyzed by PCR-based microsatellite analysis for LOH in 23 of the 29 cases. PCR primers for dinucleotide microsatellite markers were purchased from Research Genetics (Huntsville, AL) for the following loci: D22S303, D22S257, D22S301, TOP1P2, and D22S310. Radioactive end labeling, PCR, and gel analysis were performed using standards conditions, as reported previously (12).

RT-PCR.

RT-PCR was performed according to the manufacturer’s protocol (Stratagene). Two pairs of primers were designed to amplify the INI1 cDNA: INICD1.forward, 5′-CTGAGCAAGACCTTCGGGCAG-3′, and INICD1.reverse, 5′-GATGGCTGGCACAAACGTCAG-3′; and INICD2.forward, 5′-AGATCGATGGGCAGAAGCTGC-3′, and INICD2.reverse 5′-TGGAATGTGTACCGGGAAGGG-3′. The PCR conditions were as follows: initial denaturation at 94°C for 4 min, followed by 30 cycles of 94°C for 25 s, 66°C for 1 min, and 72°C for 2 min and a final extension at 72°C for 10 min. The PCR products were analyzed by electrophoresis using 2% NuSieve agarose gels (FMC). Individual bands were isolated and purified with the QIAquick gel extraction kit (Qiagen) for sequencing.

Mutation Analysis.

Oligonucleotide primers for exons 1–9 were designed from the intron/exon boundary sequences for the INI1 gene (GenBank accession nos. Y17118–Y17126). Primer sequences are available upon request. Tumor and normal DNA samples producing a PCR product for that exon were analyzed by the heteroduplex method and/or direct sequencing. Heteroduplex analysis was performed based on the method published by Kambouris et al.(13). Briefly, genomic DNA was amplified in 50-μl reaction mixtures containing 50 mm KCl, 1.5 mm MgCl2, 10 mm Tris-HCl (pH 8.3), 125 μm each dNTP, 1 unit of Taq DNA Polymerase (Boehringer Mannheim), and 25 pmol of each primer. The PCR conditions were as follows: initial denaturation at 95°C for 5 min; 30 cycles of 94°C for 30 s, 60–64°C for 30 s, and 72°C for 1 min; and a final extension at 72°C for 7 min. Heteroduplex formation was enhanced by mixing equal volumes of sample and wild-type PCR product, denaturing for 5 min at 94°C, and cooling to 37°C over a 1-h period. Five μl of this reaction mixture were combined with 1 μl of loading dye (60% sucrose, 1% bromphenol blue, and 1% xylene cyanol in distilled H2O) and subsequently electrophoresed through a vertical, 1-mm-thick 10% nondenaturing acrylamide gel containing 0.5× TTE, 15% formamide, and 10% ethylene glycol. Gels were run at room temperature in 0.5× TTE buffer for 15 h at a constant voltage of 450 V. After running, the gels were stained with a 2 μg/ml ethidium bromide solution for 5 min and destained with 0.5× TTE buffer for 10 min. The bands were then visualized using a Bio-Rad Gel Doc 1000 imaging system. To confirm any base changes observed by heteroduplex screening, the PCR products were sequenced directly.

Sequence Analysis.

Sequencing of the RT-PCR or PCR products was performed by the Nucleic Acid/Protein Core facility of The Children’s Hospital of Philadelphia. The cDNA sequence was compared to the GenBank data base (accession no. U04847) using the BLAST program through the network server at the National Center for Biotechnology. Sequence of individual exons was compared to the reported sequences in GenBank (accession nos. U04847 and Y17118–126). One apparent discrepancy compared to the GenBank sequence for U04847 was identified. Nucleotide 406, reported to be a thymidine, was found to be a cytosine in 29 patients and controls. This predicts that codon 136 will code for a proline instead of the reported serine. Sequence analysis of exon 9 in a similar set of patients was consistent with U04847 for codon 378 (CTT), not GAA (Y17126). Similarly, nucleotide 1145 in codon 382 is a cytosine instead of a guanine.

Deletion Analysis of Primary Tumors.

Clinical information for the 28 patients included in this study is shown in Table 1. There were 18 CNS AT/RTs; 7 renal rhabdoid tumors, 2 of which were from the same patient (50340-1 and -2); and 4 extrarenal (soft tissue and liver) tumors. Among the 24 cases for which age and sex were known, there were 9 females and 15 males, and patient age ranged from newborn to 7 years.

Tumor karyotypes were available for 11 patients, and are shown in Table 1. Five tumors (86-18, 88-128, 93-94, 97-54, and 98-74) demonstrated monosomy 22 as the only abnormality. Two tumors (97-102 and 96-10-P308) demonstrated unbalanced translocations of 22q11–q12 (9, 11). One tumor had a small ring chromosome 22 (97-41). Tumor 95-148 had a near-triploid karyotype with two apparently normal copies of chromosome 22. Constitutional karyotypes of patient 97-101 demonstrated a ring chromosome 22 (10).

Touch preparations from frozen tumors or cytogenetic pellets from direct preparations or 24–72-h cultures were analyzed by interphase FISH to screen for 22q11.2 deletions. Probes used for FISH included a series of cosmid clones that map to the previously defined rhabdoid tumor critical region within 22q11.2 (7).4 Tumors deleted for BCR and/or MIF(14) were considered to have a deletion of the rhabdoid tumor region. A probe for the EWS region in 22q12 was used as an internal control and to distinguish partial deletions versus monosomy 22 in the tumor cells. Among the 21 tumors analyzed by FISH, 17 demonstrated heterozygous or homozygous deletions within the rhabdoid tumor region in 22q11.2 (Table 1). Of note, analysis of lymphoblast cells from the patient with the constitutional ring chromosome 22 (97-101) demonstrated the normal number of two signals for N25 (proximal 22q11.2), BCR, and EWS but only one copy of cos82 (distal region of 22q13). FISH of touch preparations from the frozen tumor biopsy demonstrated one copy of N25, BCR, EWS, and cos82. These results were consistent with loss of the ring chromosome in the tumor.

To develop specific probes for the INI1 gene that could be used to detect deletions by FISH, we screened a chromosome 22-only cosmid library with labeled PCR products from the INI1 coding sequence. Seven clones were obtained by screening with exons 1, 4, and/or 9, and six clones were confirmed by PCR to contain the INI1-specific exon sequences (designated INI1-1 to -6). The probes were used for FISH to normal control lymphocytes, several rhabdoid tumor cell lines, and a representative series of primary rhabdoid tumors with and without detectable 22q11.2 deletions. All six INI1 probes demonstrated single hybridization signals to both copies of chromosome 22 in normal metaphase spreads. However, FISH of five of the six INI1 cosmids to interphase nuclei from normal lymphocytes, as well as fibroblasts synchronized at the G1-S boundary of the cell cycle (results not shown), demonstrated 4 signals per cell, as compared to the normal copy number of two signals per cell for the control EWS probe. One probe, INI1–4, produced a large signal but did not appear to be duplicated. Interphase analysis of the SJSC cell line, which is homozygously deleted by FISH and PCR for BCR(7) and homozygously deleted by PCR for exons 1–9 of INI1, did not demonstrate any hybridization with INI1-2 or INI1-4 but showed two signals following hybridization with the INI1-1 and INI1-3 cosmid probes. These results suggested that repeated sequences, which may be responsible for the duplication of signals, are in close proximity to the INI1 locus. Large-scale genomic sequence analysis of this region will be required to determine the nature of the repeated sequences.

Among the 11 primary rhabdoid tumors examined, six tumors demonstrated deletions with INI1-4 (Table 1; 93-58, 93-133, 97-41, 97-54, 97-55, and 96-10-P308). Furthermore, cosmids INI1-2 and INI1-4 were homozygously deleted in case 95-148, whereas INI1-1, -3, and -5 were present in at least two copies. These results also suggest that the repeat sequences are in close proximity to each other. The FISH results for 95-148 were consistent with the PCR studies of the genomic DNA from that tumor, which demonstrated a homozygous deletion of exon 1 (Table 2). The FISH studies with the INI1 cosmid probes for four other primary tumors were equivocal, and the data are therefore not presented.

DNA from 23 tumors and matched normal tissue (when available) was screened by PCR-based microsatellite analysis to identify additional deletions in the rhabdoid tumor region between BCR and INI1 (Table 1). Representative results are shown in Fig. 1. Nineteen tumors demonstrated apparent LOH with one or more of the following markers: D22S303, D22S257, D22S301, and TOP1P2, which map between the immunoglobulin loci and the region distal to INI1 in 22q11. Interestingly, at least two tumors (50340-2 and 50495) demonstrated LOH but did not appear to be deleted by FISH. These results are consistent with loss and reduplication or mitotic recombination of all or part of chromosome 22 as a mechanism for generating LOH.

Combining the karyotype, FISH, and PCR-based microsatellite analysis, a total of 16 of 18 brain tumors, 3 of 7 renal tumors, and 4 of 4 extrarenal tumors demonstrated heterozygous or homozygous deletions in the BCR to INI1 region of chromosome 22q11.2.

Expression and Sequence Analysis of Primary Rhabdoid Tumors by RT-PCR.

Frozen tumor tissue was available for isolation of total RNA and RT-PCR studies for seven of the primary tumors. Expression of the 5′ (exons 1–6) and 3′ (exons 5–9) portions of the INI1 cDNA was observed for three of the seven tumors, as shown in Fig. 2. Sequence analysis of the three tumors was performed to identify possible mutations. As shown in Table 2, the kidney tumor 98-126 contained a C-to-G transition at nucleotide 727 of the cDNA (codon 243) in exon 6, which changes a glutamine to a stop codon. The brain tumor from this patient was not biopsied. Tumor 50462-1 contained a G-to-A mutation at nucleotide 617 (exon 5, codon 206), which predicts translation of a stop codon instead of a tryptophan. The mutation was heterozygous in the RNA and DNA of the tumor and was also present in matched DNA from the patient’s blood. We have not yet detected an alteration in the other allele. Analysis of genomic DNA from the third tumor, E411, demonstrated a heterozygous C472T mutation in exon 4 (see below).

Heteroduplex and Sequence Analysis of Genomic DNA from Primary Tumors.

PCR analysis of genomic DNA using primers designed from the intron/exon boundary regions for each of the nine exons was first performed to identify intragenic deletions. Two CNS AT/RTs (93-58 and 97-102) and two extrarenal rhabdoid tumors (97-55 and 96-10-P308) demonstrated homozygous deletions of all nine exons. Ten tumors (7 CNS, 1 renal, and 2 extrarenal) contained homozygous deletions of exon 1. PCR of exons 2–9 indicated the presence of at least one copy of each of these exons, placing the distal breakpoint of the deletions between the 5′ untranslated region and the 3′ region of intron 1. In four cases, we could show that the homozygous deletions were present in the tumor only because matched DNA from blood samples contained at least one copy of exon 1 (Fig. 3). These results may explain the lack of expression of INI1 observed for cases 95-148, 93-94, and 98-69, as shown in Fig. 2.

As an initial means to screen for mutations, heteroduplex analysis of 15 DNA samples was performed using primers for exons 2–6 and exon 8. Bandshifts were observed (data not shown) for two tumors for exon 4 (88-128 and E411), two tumors for exon 5 (93-68 and 95-46), and three tumors (86-18, 98-126, and 50495) for exon 6. Sequence analysis of tumor DNA for the exons producing bandshifts demonstrated single bp changes predicted to change an amino acid to a stop codon (Table 2). The C727T mutation in 98-126, initially identified by sequence analysis of the RNA, was confirmed in the tumor DNA. Sequence analysis of the matched blood DNA demonstrated the normal sequence. Case E411 contained a heterozygous C-to-T mutation at nucleotide 472 (exon 4, codon 158), which changes an arginine to a stop codon. The same heterozygous mutation was present in the constitutional DNA, consistent with a germ-line mutation. Sequence analysis of exon 4 in the parental DNA samples was normal, suggesting that the mutation was not inherited. It is not known whether the normal allele detected by the sequence analysis is due to normal contamination within the tumor or whether there is a second mutation in the other allele.

On the basis of a relatively low frequency of mutations detected by the initial heteroduplex analysis, we directly sequenced the remaining tumors for possible mutations. As shown in Table 2, mutations in five additional patients were identified by direct sequencing. Case 98-74 demonstrated a G-to-A change in nucleotide 152 (codon 51) in exon 2, which changes a tryptophan to a stop codon. There was no evidence for a second allele by the sequence analysis, consistent with cytogenetic studies that had shown monosomy 22 in the tumor cells. The matched blood specimen demonstrated the same mutation, as well as the presence of the normal allele. Interestingly, expression of the INI1 gene was not readily detected by RT-PCR in this tumor, which cannot be explained by this mutation.

Sequence analysis of AT/RT 97-101 demonstrated a 4-bp insertion at codon 46 (exon 2), causing a frameshift. The constitutional ring chromosome in this patient was deleted in the tumor, so that the mutation must have been in the other homologue. Analysis of the lymphoblastoid DNA demonstrated only the normal sequence for exon 2, and thus, the insertion mutation was a somatic event. Frameshift mutations were detected in two other brain tumors, 93-133 and 94-90, each of which contained a 1-bp deletion in codon 382 or 383 in exon 9.

Patient 50340 had bilateral renal rhabdoid tumors, both of which demonstrated the same 10-bp deletion in exon 7. Sequence analysis of matched normal tissue from this patient showed that the deletion was also present in the germ line. DNA from the blood and one of the tumors was analyzed by PCR-based microsatellite analysis and demonstrated LOH for D22S257. There was no apparent deletion by FISH with cosmid probes for the BCR or INI1 genes, suggesting loss and reduplication or mitotic recombination leading to the LOH. Finally, a C-to-T mutation in the renal rhabdoid tumor 50495 was identified at base 727, predicted to change a glutamine to stop codon.

We analyzed a large series of 29 AT/RTs of the brain, kidney, liver, and soft tissues and demonstrate that deletions and/or mutations of the INI1 gene are present in all cases. The data support our hypothesis that tumors of similar histological appearance with overlapping deletions of 22q11.2 may result from homozygous inactivation of the same gene and may present at any site. Furthermore, observation of germ-line mutations within the coding sequence of INI1, accompanied by somatic loss or mutation of the remaining allele, supports the model of a tumor suppressor gene.

AT/RTs account for ∼2% of pediatric brain tumors, although the incidence may increase as pediatric neuropathologists become more adept at distinguishing AT/RT from MB/PNET. MB and PNET are the most common malignant brain tumors in children, accounting for 25% of childhood CNS malignancies. Both PNETs and AT/RTs have complex histological patterns, and because two-thirds of ATTs have areas of primitive neuroepithelial tissue, the AT/RTs need to be distinguished from “pure” PNETs by careful study of the histology and use of a series of antibodies to epithelial, connective tissue, and neural markers. In contrast to the almost uniformly fatal outcome for children with AT/RT, as well as rhabdoid tumors arising in other sites, combined modality treatment including surgery, radiation, and chemotherapy has produced 5-year survival rates that approach 70–80% (15) for MB/PNET. Nevertheless, young age at diagnosis has been considered a poor prognostic factor for children with MB/PNET. We have suggested that children younger than 2 years of age whose PNET is really an AT/RT may account for the poor survival in this age group.

It is possible that inactivation of INI1 may be associated with a poor prognosis, even if the rhabdoid component of the tumor is not apparent by histological or immunological studies. An example of this is evidenced by the results of the INI1 deletion analysis for patient 93-94. A small specimen from this 1-year-old girl was diagnosed by the institutional pathologist as well as two independent neuropathology reviewers as a MB/PNET. This could represent a sampling problem because cytogenetic studies demonstrated monosomy 22 as the only detectable abnormality, and interphase FISH analysis confirmed the loss of one copy of the BCR gene in 22q11.2. As shown in Table 2, the tumor contained a homozygous deletion in exon 1 of the INI1 gene and absent expression by RT-PCR studies (Fig. 2), consistent with homozygous inactivation of the gene. Furthermore, this patient had a recurrence and subsequently died 1 year and 2 months from the time of diagnosis. It is clear that the identification of genetic alterations of INI1 will be important in diagnosis and for determination of prognosis for patients with these histologically complex malignancies.

Among the 29 CNS AT/RT, renal, and extrarenal rhabdoid tumors that we have reported here, the frequency of deletion of the entire coding sequence of the INI1 gene appears to be lower than the 6 of 13 malignant rhabdoid tumors reported by Versteege et al.(8). Only 5 of the 29 tumors were homozygously deleted for the INI1 gene, as determined by FISH or PCR. However, it was surprising that 10 of these 29 tumors demonstrated homozygous deletions in exon 1. In cases in which normal tissue was available for study, it was clear that at least one copy of exon 1 was present in the germ line; however, we have not yet determined whether one or two copies of the exon 1 region are present in the lymphocyte DNA. Deletion of exon 1 would predispose individuals to the development of rhabdoid tumors, and thus, the number of germ-line mutations observed in this series of patients may be an underestimate of the actual frequency.

It is noteworthy that similar deletions, especially those involving exon 1, were observed in the brain, renal, and extrarenal tumors, and thus, the mechanisms leading to deletion may be sequence related. In fact, the 22q11.2 region may be more prone to deletions because of the repeat sequences in this part of the chromosome. The interphase FISH studies with the INI1 cosmid probes suggest that a repeat sequence is present in the INI1 gene and/or that the INI1 gene itself is duplicated. The latter is less likely, based on the small number of positive clones identified by the cosmid library screen and the ability to detect homozygous deletions of exons within the gene in tumor DNA. Evidence for loss and reduplication or mitotic recombination within 22q11.2 was supported by LOH for microsatellite markers between BCR and TOP1P2, in the presence of the normal copy number of probes for the region as detected by FISH. These results may also reflect the instability of this region of the chromosome and may account for the relatively high frequency of deletions.

Like deletions, mutations observed for the AT/RTs do not appear to differ from the mutations identified in the renal and other extrarenal rhabdoid tumors. We documented frameshifts due to an insertion or deletion in three brain tumors as well as the bilateral kidney tumors from patient 50340. Similarly, nine nonsense mutations were observed in five CNS tumors and four kidney tumors. Three of the germ-line mutations, in one AT/RT and two renal rhabdoid tumor patients, were single bp substitutions predicted to cause premature truncation of the protein. In contrast, the germ-line mutation in the patient with bilateral renal tumors was a 10-bp deletion, which results in a frameshift mutation and predicted stop codon from the TGA beginning at nucleotide 884 in exon 7. Additional studies are required to determine how specific mutations may predispose children to CNS, renal, or extrarenal rhabdoid tumors.

INI1 is the smallest member of the human SWI/SNF complex, which is currently known to contain at least 9–12 proteins (16). The SWI/SNF complex is thought to function in an ATP-dependent manner to cause a conformational change in the nucleosome that alters histone-DNA binding (17), thereby facilitating transcription factor access. Studies of the mammalian SWI/SNF complex have suggested that different cells may contain distinct subunits and, thus, may function to remodel chromatin in a cell-specific manner (16). INI1, however, is thought to be a component of most complexes. INI1 was also isolated through its interaction with HIV integrase and is thought to mediate retroviral integration of HIV into the genome (18).

Studies of the INI1 Drosophila homologue, snr1, have shown that snr1 is expressed at high levels during early embryogenesis and, in later development, is expressed most highly in the CNS (19). snr1 homozygous mutants die during embryogenesis, also supporting its role as a tumor suppressor gene. Dingwall et al.(19) reported that snr1 interacts with trithorax, an activator of homeotic gene transcription. Rosenblatt-Rosen et al.(20) have shown recently that the human homologue of trithorax, ALL-1, also interacts with INI1 and proposed that the SWI/SNF complex is recruited to ALL-1 target genes through its interaction with INI1. The trithorax-polycomb group of proteins controls expression of a variety of homeotic genes required for normal development. In humans, chromosomal translocation of ALL-1 (MLL) with a variety of partners results in a chimeric fusion protein, expression of which is likely a major initiating event in acute leukemia, particularly in infants. These studies provide further support for a role of INI1 in both normal development, as well as malignant transformation.

As shown in Fig. 4, the mutations identified in INI1 are distributed throughout the coding sequence. Interestingly, one brain and one kidney tumor had the same mutation in base 472 of exon 4, and two brain tumors had similar 1-bp deletions within exon 9, suggesting that there may be hotspots for specific mutations. Seven of the mutations are located in the three highly conserved regions of the protein. The three conserved regions between the INI1, snr1 (Drosophila), CeSNF5 (Caenorhabditis elegans), and yeast SNF5 proteins (21), include two repeat motifs, the first of which is required for binding to the brg-1 protein, a major component of the SWI/SNF complex (22). Each mutation is predicted to code for a stop codon and is likely to alter or prevent binding of INI1 in the complex. Brg-1 has been shown to alter cell growth, and it interacts with the hypophosphorylated retinoblastoma protein, thus implicating a pathway in which INI1 may affect the cell cycle (23).

These studies suggest that germ-line mutations in the tumor suppressor gene INI1 predispose children to the development of rhabdoid tumors. The finding of acquired mutations in all of the histologically confirmed tumors analyzed to date, as well as in a putative PNET of the brain, suggest that molecular genetic studies will have diagnostic utility in both a genetic and oncological setting.

Fig. 1.

LOH for markers within 22q11.2 in five rhabdoid tumors. Normal DNA (left) and tumor DNA (right) are shown for each tumor. Loss of an allele is shown for D22S303 in tumor 91-23; D22S257 in tumors 95-46, 97-78, and 50495; and TOP1P2 in tumor 95-148.

Fig. 1.

LOH for markers within 22q11.2 in five rhabdoid tumors. Normal DNA (left) and tumor DNA (right) are shown for each tumor. Loss of an allele is shown for D22S303 in tumor 91-23; D22S257 in tumors 95-46, 97-78, and 50495; and TOP1P2 in tumor 95-148.

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Fig. 2.

Expression of the INI1 gene in seven primary rhabdoid tumors. RT-PCR of the 5′ region (top) and 3′ region (middle) of the gene amplified a product in three tumors, shown in Lanes 3, 6, and 7. The lower band in the top panel reflects the alternatively spliced transcript, due to a cryptic splice site in exon 2 (21). RT-PCR of the same cDNA samples for glyceraldehyde 3-phosphate dehydrogenase (G3PDH; bottom) is shown for comparison. Lane 1, 95-148; Lane 2, 93-94; Lane 3, 98-126; Lane 4, 98-69; Lane 5, 98-74; Lane 6, 50462; Lane 7, E411.

Fig. 2.

Expression of the INI1 gene in seven primary rhabdoid tumors. RT-PCR of the 5′ region (top) and 3′ region (middle) of the gene amplified a product in three tumors, shown in Lanes 3, 6, and 7. The lower band in the top panel reflects the alternatively spliced transcript, due to a cryptic splice site in exon 2 (21). RT-PCR of the same cDNA samples for glyceraldehyde 3-phosphate dehydrogenase (G3PDH; bottom) is shown for comparison. Lane 1, 95-148; Lane 2, 93-94; Lane 3, 98-126; Lane 4, 98-69; Lane 5, 98-74; Lane 6, 50462; Lane 7, E411.

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Fig. 3.

Homozygous deletions of exon 1 in primary rhabdoid tumors detected by PCR. Representative results from 6 of the 10 cases are shown. Lanes B, normal DNA isolated from blood; Lanes T, DNA isolated from primary tumor tissue. Matched blood DNA was not available for tumor 97-104.

Fig. 3.

Homozygous deletions of exon 1 in primary rhabdoid tumors detected by PCR. Representative results from 6 of the 10 cases are shown. Lanes B, normal DNA isolated from blood; Lanes T, DNA isolated from primary tumor tissue. Matched blood DNA was not available for tumor 97-104.

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Fig. 4.

Schematic of the INI1 coding sequence, demonstrating the regions of conserved homology with the yeast and Drosophila SNF5 proteins. , region I; ▪, region II; , region III (21). Locations of the deletions and mutations observed in the 24 rhabdoid tumors are shown (arrows). Homozygous deletions of exon 1 were observed in 10 tumors.

Fig. 4.

Schematic of the INI1 coding sequence, demonstrating the regions of conserved homology with the yeast and Drosophila SNF5 proteins. , region I; ▪, region II; , region III (21). Locations of the deletions and mutations observed in the 24 rhabdoid tumors are shown (arrows). Homozygous deletions of exon 1 were observed in 10 tumors.

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The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1

Supported by National Cancer Institute Grant CA46274, the W. W. Smith Charitable Trust, and the Collin Schoberg Foundation.

3

The abbreviations used are: CNS, central nervous system; PNET, primitive neuroectodermal tumor; ATT, atypical teratoid tumor; AT/RT, atypical teratoid and rhabdoid tumor; MB, medulloblastoma; FISH, fluorescence in situ hybridization; LOH, loss of heterozygosity; RT-PCR, reverse transcription-PCR.

4

Unpublished data.

Table 1

Clinical findings in 29 AT/RTs

Patient no.AgeSexLocationDiagnosisKaryotypeFISHLOH
86-18 6 months Brain, kidney Rhabdoid 45,XY,−22 NAa 2 (D22S257) 
88-128 11 months Cerebellum AT/RT 45,XY,−22 NA LOH (D22S303, TOP1P2
91-23 3 yr Right frontal lobe AT/RT 46,XY HD (MIFLOH (D22S303, TOP1P2
93-58 10 months Cerebellopontine angle AT/RT NA HD (INI1LOH (D22S303) 
93-68 2 yr Right temporal parietal AT/RT NA NA 1 (D22S303, TOP1P2
93-94 1 yr Cerebellum PNET 45,XX,−22 D (BCRLOH (D22S303) 
93-133 18 months Cerebellar vermis AT/RT NA D (INI1LOH (D22S257) 
94-90 17 months Cerebellopontine angle AT/RT NA D (BCRLOH (D22S257, D22S310
94-121 5 months Cerebellum AT/RT NA D (BCRH (D22S310) 
95-46 13 months Thalamus AT/RT 46,XY D (MIFLOH (D22S257, D22S310
95-148 6 yr Left frontal lobe AT/RT 61,XX,+1,+1,+7,+7,+11,+11,+14,+14,add(16q?),+19,+add(19)(q13),+20,+20,+21,+mar,+mar HD (5′INI1LOH (TOP1P2) 
96-79 20 months Pineal AT/RT NA NA H (TOP1P2) 
97-41 22 months Brain AT/RT 46,XY,r(22) HD (INI11 (D22S303, D22S257
97-54 5 yr Left frontal lobe AT/RT 45,XY,−22 D(BCR, INI1LOH (D22S257, TOP1P2
97-55 Birth Liver, lymph nodes, leg Rhabdoid NA HD (BCR, INI11 (D22S257) 
97-64 1 week Submandibular, thyroid glands Rhabdoid NA D (BCR1 (D22S303, D22S257
97-78 3 yr Liver Rhabdoid NA D (BCR, EWSLOH (D22S303, D22S257
97-101 4 yr Third ventricle AT/RT 46,XX,r(22) (blood) D (BCR, EWSNA 
97-102 7 yr Right parietal, parasagittal AT/RT 45,XY,−22,der(22)t(22;22) HD (MIF1 (D22S303, D22S257
      D (EWS 
97-104 6 yr Left temporal lobe AT/RT NA NA H (D22S257) 
98-69 8 months Kidney, brain Rhabdoid NA ND (MIFNA 
98-74 6 months Right intraventricular AT/RT 45,XX,−22 NA NA 
98-126 17 months Kidney Rhabdoid NA D (MIFNA 
96-10-P308 3 weeks Skin, bone, soft tissue Rhabdoid 46,XY,t(10;16)(q23.2;q12), add(22) HD (INI11 (D22S303) 
50340-1   Bilateral, kidney Rhabdoid NA NA NA 
50340-2   Bilateral, kidney Rhabdoid NA ND (BCRLOH (D22S257) 
50495   Kidney Rhabdoid NA ND (BCRLOH (D22S257) 
50462-1   Kidney Rhabdoid NA NA NA 
E411   Kidney Rhabdoid NA ND (BCR1 (TOP1P2) 
Patient no.AgeSexLocationDiagnosisKaryotypeFISHLOH
86-18 6 months Brain, kidney Rhabdoid 45,XY,−22 NAa 2 (D22S257) 
88-128 11 months Cerebellum AT/RT 45,XY,−22 NA LOH (D22S303, TOP1P2
91-23 3 yr Right frontal lobe AT/RT 46,XY HD (MIFLOH (D22S303, TOP1P2
93-58 10 months Cerebellopontine angle AT/RT NA HD (INI1LOH (D22S303) 
93-68 2 yr Right temporal parietal AT/RT NA NA 1 (D22S303, TOP1P2
93-94 1 yr Cerebellum PNET 45,XX,−22 D (BCRLOH (D22S303) 
93-133 18 months Cerebellar vermis AT/RT NA D (INI1LOH (D22S257) 
94-90 17 months Cerebellopontine angle AT/RT NA D (BCRLOH (D22S257, D22S310
94-121 5 months Cerebellum AT/RT NA D (BCRH (D22S310) 
95-46 13 months Thalamus AT/RT 46,XY D (MIFLOH (D22S257, D22S310
95-148 6 yr Left frontal lobe AT/RT 61,XX,+1,+1,+7,+7,+11,+11,+14,+14,add(16q?),+19,+add(19)(q13),+20,+20,+21,+mar,+mar HD (5′INI1LOH (TOP1P2) 
96-79 20 months Pineal AT/RT NA NA H (TOP1P2) 
97-41 22 months Brain AT/RT 46,XY,r(22) HD (INI11 (D22S303, D22S257
97-54 5 yr Left frontal lobe AT/RT 45,XY,−22 D(BCR, INI1LOH (D22S257, TOP1P2
97-55 Birth Liver, lymph nodes, leg Rhabdoid NA HD (BCR, INI11 (D22S257) 
97-64 1 week Submandibular, thyroid glands Rhabdoid NA D (BCR1 (D22S303, D22S257
97-78 3 yr Liver Rhabdoid NA D (BCR, EWSLOH (D22S303, D22S257
97-101 4 yr Third ventricle AT/RT 46,XX,r(22) (blood) D (BCR, EWSNA 
97-102 7 yr Right parietal, parasagittal AT/RT 45,XY,−22,der(22)t(22;22) HD (MIF1 (D22S303, D22S257
      D (EWS 
97-104 6 yr Left temporal lobe AT/RT NA NA H (D22S257) 
98-69 8 months Kidney, brain Rhabdoid NA ND (MIFNA 
98-74 6 months Right intraventricular AT/RT 45,XX,−22 NA NA 
98-126 17 months Kidney Rhabdoid NA D (MIFNA 
96-10-P308 3 weeks Skin, bone, soft tissue Rhabdoid 46,XY,t(10;16)(q23.2;q12), add(22) HD (INI11 (D22S303) 
50340-1   Bilateral, kidney Rhabdoid NA NA NA 
50340-2   Bilateral, kidney Rhabdoid NA ND (BCRLOH (D22S257) 
50495   Kidney Rhabdoid NA ND (BCRLOH (D22S257) 
50462-1   Kidney Rhabdoid NA NA NA 
E411   Kidney Rhabdoid NA ND (BCR1 (TOP1P2) 
a

NA, not available or not adequate; D, deletion; ND, no deletion; H, heterozygous; 1, one allele present; 2, two alleles present; HD, homozygous deletion.

Table 2

INI1 mutations in AT/RTs

Patient no.LocationMutationExon(s)CodonPredicted effect
86-18 Brain C778T 260 Gln→Stop 
88-128 Brain C472T 158 Arg→Stop 
91-23 Brain Homozygous deletion  Altered or absent expression 
93-58 Brain Homozygous deletion 1–9  No protein 
93-68 Brain C601T 201 Arg→Stop 
93-94 Brain Homozygous deletion  Altered or absent expression 
93-133 Brain delC 382 or 383 Frameshift 
94-90 Brain delC 382 or 383 Frameshift 
94-121 Brain Homozygous deletion  Altered or absent expression 
95-46 Brain G618A 206 Trp→Stop 
95-148 Brain Homozygous deletion  Altered or absent expression 
96-79 Brain Homozygous deletion  Altered or absent expression 
97-41 Brain Homozygous deletion 1–9  No protein 
97-54 Brain Homozygous deletion  Altered or absent expression 
97-55 Inguinal mass Homozygous deletion 1–9  No protein 
97-64 Neck mass Homozygous deletion  Absent or altered expression 
97-78 Liver Homozygous deletion  Absent or altered expression 
97-101 Brain 138ins TACC 46 Frameshift 
97-102 Brain Homozygous deletion 1–9  No protein 
97-104 Brain Homozygous deletion  Absent or altered expression 
98-69 Kidney Homozygous deletion  Absent or altered expression 
98-74 Brain, blood G152A 51 Trp→Stop 
98-126 Kidney C727T 243 Gln→Stop 
96-10-P308 Abdominal nodule Homozygous deletion 1–9  No protein 
50340-1 Kidney, blood 797del 10 bases 266 Frameshift 
50340-2 Kidney 797del 10 bases 266 Frameshift 
50495 Kidney C727T 243 Gln→Stop 
50462-1 Kidney, blood G617A 206 Trp→Stop 
E411 Kidney, blood C472T 158 Arg→Stop 
Patient no.LocationMutationExon(s)CodonPredicted effect
86-18 Brain C778T 260 Gln→Stop 
88-128 Brain C472T 158 Arg→Stop 
91-23 Brain Homozygous deletion  Altered or absent expression 
93-58 Brain Homozygous deletion 1–9  No protein 
93-68 Brain C601T 201 Arg→Stop 
93-94 Brain Homozygous deletion  Altered or absent expression 
93-133 Brain delC 382 or 383 Frameshift 
94-90 Brain delC 382 or 383 Frameshift 
94-121 Brain Homozygous deletion  Altered or absent expression 
95-46 Brain G618A 206 Trp→Stop 
95-148 Brain Homozygous deletion  Altered or absent expression 
96-79 Brain Homozygous deletion  Altered or absent expression 
97-41 Brain Homozygous deletion 1–9  No protein 
97-54 Brain Homozygous deletion  Altered or absent expression 
97-55 Inguinal mass Homozygous deletion 1–9  No protein 
97-64 Neck mass Homozygous deletion  Absent or altered expression 
97-78 Liver Homozygous deletion  Absent or altered expression 
97-101 Brain 138ins TACC 46 Frameshift 
97-102 Brain Homozygous deletion 1–9  No protein 
97-104 Brain Homozygous deletion  Absent or altered expression 
98-69 Kidney Homozygous deletion  Absent or altered expression 
98-74 Brain, blood G152A 51 Trp→Stop 
98-126 Kidney C727T 243 Gln→Stop 
96-10-P308 Abdominal nodule Homozygous deletion 1–9  No protein 
50340-1 Kidney, blood 797del 10 bases 266 Frameshift 
50340-2 Kidney 797del 10 bases 266 Frameshift 
50495 Kidney C727T 243 Gln→Stop 
50462-1 Kidney, blood G617A 206 Trp→Stop 
E411 Kidney, blood C472T 158 Arg→Stop 

We acknowledge the support of the National Wilms’ Tumor Study Group and Cooperative Human Tissue Network for providing tissue and DNA. We are grateful to the following colleagues for providing clinical information and samples or performing pathology review: Leslie Sutton, Kathy Bonner, Ann-Christine Duhaime, Katrina Conard, Rita Meek, Joanne Hilden, Carolyn Russo, Anne Bendel, Jeanette Camacho, Carl Lenarsky, Doug Miller, James Powers, Mary Davis, Debbie Belchis, Jeffrey Golden, Margaret Collins, Michael Willoughby, James Boyett, and Paul Grundy, as well as participating investigators within the Children’s Cancer Group. We thank Annette Parmiter, Deborah Kramer, and Zhili Wang for technical support and suggestions and Regina Harvey for manuscript preparation.

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