The 100 cases included 65 patients with CNS AT/RT, 12 with MRT of the kidney, 17 with extra-renal tumors, and 6 patients with multiple primary tumors (, , and ). There were 49 males and 51 females. As shown in , and , 35 of 100 patients had a germline deletion or mutation. Patients with a germline abnormality ranged from birth to 5 years with a median age at diagnosis of 5 months. The age at diagnosis for patients with sporadic tumors (only somatic mutations/deletions detected) ranged from birth to 17 years with a median age of 18 months. Five of the 65 patients with sporadic tumors were diagnosed over 6 years of age, and two of these were teenagers.
SMARcb1 Alterations in 35 Patients With Germline Abnormalities Predisposing to Rhabdoid Tumor
SMARCB1 Alterations in 65 Patients With Rhabdoid Tumor Caused by Somatic Abnormalities
Germline Alterations in Rhabdoid Tumors by Anatomic Site
Fig 1 Germline alterations were found in 35 cases and included mutations, duplications and deletions (shown in black). The “second hit,” found only in tumor cells, is shown in white. The last seven patients (01-135, 07-315, 00-214, 09-289, 06-147, (more ...)
As was expected, patients with multiple tumors were most likely to have a germline mutation. Six of 6 patients with two primary tumors, as assessed by clinical presentation and/or distinct molecular alterations as the second inactivating event, had a germline mutation. Twenty-three of 65 patients with CNS tumors had a germline mutation, compared with 3 of 12 patients with renal tumors and 3 of 17 patients with soft tissue tumors (). In contrast, there was a high frequency (9 of 17 cases) of somatic homozygous whole gene deletions in tumors from extra-renal sites, as previously reported [19
The most common germline abnormalities detected were point or frameshift mutations resulting in premature truncation of the protein (14 of 35 cases). Other germline abnormalities included heterozygous loss of SMARCB1 (9 of 35 cases), and duplications (3 of 35 cases) or deletions (8 of 35 cases) of one or more exons within SMARCB1 (). All of the germline deletions of 22q11.2 that included the entire SMARCB1 locus appeared to be de novo. There was one family with a splice site mutation (04–064). A mutation in exon 4 (c.472C>T) was demonstrated in two cases. All of these abnormalities were also found in patients with somatic alterations of SMARCB1. The second inactivating event in the tumors from these patients was a deletion (16), copy number neutral loss of heterozygosity (CN LOH) (10 tumors), or a frameshift mutation (8). There was only one tumor with a point mutation as the second hit (case 07–158).
Both parents' blood samples were available for testing in 22 of the 35 cases with germline mutations. In 7 of these 22 cases a parent was a carrier of the germline abnormality, four of which were maternally inherited and 3 of which were paternally inherited (). Two of the carrier fathers (cases 06–165 and 07–367) developed schwannomas (). The mother of patient 08–143 was diagnosed with a benign CNS lesion (). The other 4 carrier parents were unaffected. In two additional cases (07-005 and 09–013) the parents' blood samples were normal, but there were two affected siblings with the same mutation, consistent with gonadal mosaicism ().
Fig. 3 Pedigree of family 09-013. Two affected sisters had the same SMARCB1 exon 1 mutation (c.20_43delinsT). Neither parent had the mutation, consistent with gonadal mosaicism (A). Pedigree of family 07-367. The proband (III.1) was found to have a germline (more ...)
The deletions and duplications within SMARCB1
encompassed anywhere from 1 exon to all 9 exons (). These small changes in copy number preferentially included exons 4 and 5, which seem to be a particular hot spot for both germline and somatic abnormalities. Duplications and deletions proximal and distal to SMARCB1
were identified by both MLPA and SNP array analyses (data not shown). The largest germline deletions detected by MLPA all spanned from 22q11.21–q11.23 and included MAPK1
(6 of 9 cases). Two of these children presented with phenotypic abnormalities, and were analyzed by SNP array. All genomic positions were based upon hg18 (March, 2006) from the UCSC Genome Browser (http://genome.ucsc.edu
). Case 07–158 demonstrated a de novo
deletion from 20,247,190 to 22,973,609. He presented with several phenotypic defects including ptosis, small cupped auricular deformity, and pectus excavatum. He also had a small ventricular septal defect. Case 08–092 showed a de novo
deletion spanning from 20,128,907 to 23,011,579. He was noted to have a patent foramen ovale, a slightly upslanting palpebral fissure and his ears were low set with posterior rotation. We previously reported similar phenotypic findings in rhabdoid patients with germline deletions proximal to, and including, SMARCB1
In one case (08–237) there was a germline deletion of GNAZ in chromosome band 22q11.22, proximal to SMARCB1 (). The tumor demonstrated loss of the deleted chromosome, as well as a mutation in the remaining copy of SMARCB1. As the GNAZ deletion was also found in the proband's unaffected brother and mother, it was not considered to be the predisposing rhabdoid tumor deletion. However, there may be some underlying genetic instability in these individuals that resulted in multiple alterations of chromosome 22 in the affected child.
Fig. 2 Illumina Beadstudio depiction of chromosome 22 showing a heterozygous deletion in the blood of patient 08-237. A decrease in the Log R ratio corresponding to the absence of heterozygous SNPs indicates a deletion, the location of which is demonstrated (more ...)
In two cases, mutations were found in the splice sites of exons. In one family (case 04–064) three of four children were diagnosed with AT/RT and showed an intron 7 point mutation (c.986+1G>C) in the donor splice site (). Sequence analysis of a RT-PCR product spanning exons 5 to 9 demonstrated that the splice site mutation resulted in the exclusion of exon 7. A similar mutation in the donor splice site of exon 7 was previously reported in a family with a history of posterior fossa brain tumors in infancy [20
]. Both parents were screened and the father was found to carry the same mutation as the affected children. The paternal grandmother and aunts were also tested and found to be normal. Clinical details of this case have been published separately [21
]. The second case (09–019) had a somatic mutation in the acceptor splice site of exon 5 (c.501−1G>C) and CN LOH of the other allele that spanned from 22q11.21 to the terminus.
There were seven patients in the present study that had at least one affected sibling. The pedigree shown in , case 09–013, represents a family with two affected daughters. The first daughter was diagnosed at 5 months with a bladder mass, reported as a poorly differentiated sarcoma. The second daughter developed a brain tumor at 2 months of age. This tumor was diagnosed as an AT/RT, and SMARCB1 mutation screening was performed. Sequence analysis revealed a mutation in exon 1 in the blood and tumor (c.20_43delinsT) (). MLPA analysis was consistent with a deletion of the second allele. Formalin fixed tissue preserved from the older daughter's tumor was then tested and found to have the same mutation. MLPA analysis of both of their tumors showed a deletion of probe E1b, which overlaps the mutated region of exon 1. The sequencing and MLPA studies were normal in the peripheral blood DNA from both parents. Results from the SNP array studies confirmed the paternity and there were no significant abnormalities found in either parent. Gonadal mosaicism is therefore the most likely explanation for the inheritance of the germline mutation seen in these two siblings.
A second family (case 07-005) in our study population showed a similar pattern of inheritance. This family presented with two children with rhabdoid tumors, a male and female. Both siblings showed a duplication of SMARCB1
exons 4–5 by MLPA (). The MLPA results from the parents' blood DNA indicated normal SMARCB1
copy number. These results were confirmed with a TaqMan assay designed specifically to reveal copy number of SMARCB1
exons 4–5 (data not shown). The SNP array confirmed paternity and did not reveal any significant abnormalities. Details of this family have been published separately [21
The pedigree shown in represents the second family to be reported in which a germline mutation predisposed to both schwannomatosis and CNS AT/RT. The proband (III.1) was diagnosed with an AT/RT. Testing of peripheral blood DNA was prompted due to the identification of an exon 4 (c.472C>T) mutation in the tumor, as this frequently presents as a germline mutation (). The paternal grandmother also had a history of multiple schwannomas. The father and grandmother were both found to be carriers. Although the grandmother's brother (I.3) was reported to have died from medulloblastoma, a diagnosis of AT/RT is more likely, given that there is a SMARCB1 mutation segregating in the family. The patient's father (II.2) was subsequently found to have a schwannoma. Prenatal testing for III.2 was performed, and there was no mutation identified.
The third pedigree () depicts a family in which the son was initially reported to have an epithelioid malignant peripheral nerve sheath tumor. His sister was subsequently diagnosed with a CNS AT/RT, prompting genetic testing and pathologic review of the son's tumor. The tumor from III.1 had the same mutation (c.367C>T) found in the tumor and blood from his sister (III.2) and was ultimately re-classified as MRT. The mother was found to carry the same exon 4 mutation. Extensive testing of her parents and siblings suggested that this was a de novo mutation. She was later found to have an ill-defined CNS lesion, not suggestive of AT/RT, with retained SMARCB1.
There were 65 of the 100 cases in which both inactivating SMARCB1
events were somatic in origin (). These abnormalities included 89 deletions in 26 patients, 7 intragenic deletions in 7 patients, 22 frameshift mutations in 20 patients, and 12 point mutations in 12 patients. The most common finding was a homozygous deletion (26 of 65 tumors). The deletions were approximately the same size as the deletions in the germline associated cases. The most common mutation was a frameshift (16 tumors) in exon 9, either c.1143delG or c.1145delC, both of which are predicted to cause an elongation of the protein by 104 amino acids. The protein in these tumors is not expressed, since immunohistochemical analysis always demonstrates loss of expression in tumor nuclei [22
]. These single base deletions are the most common mutations observed in CNS AT/RTs and yet have never been seen in the germline. To date, they have not been reported in renal or extra-renal malignancies. Whether this could be due to some dominant negative effect in the germline is not known. As in the germline cases, an exon 4 mutation, c.472C>T, was seen multiple times (3 of 12 point mutations). An exon 5 mutation, c.601C>T, was the most common point mutation observed (4 of 12 point mutations).
A total of 23 trios (patients and parents) were analyzed to determine the parental origins of chromosomes harboring the SMARCB1 abnormalities (11 sporadic tumors and 12 germline associated tumors). Overall, of 15 mutations, 7 were found on the paternal allele and 8 were found on the maternal allele. Of 4 intragenic deletions, 1 was on the paternal allele, and 3 were on the maternal allele. Among 27 deletions, 15 were on the paternal allele and 12 were on the maternal allele. These data do not seem to show a bias in the parental origin of the abnormality; however, further studies are required in order to draw any significant conclusions.