Evidence of a four-hit mechanism involving SMARCB1 and NF2 in schwannomatosis-associated schwannomas

Schwannomatosis is characterized by the onset of multiple intracranial, spinal, or peripheral schwannomas, without involvement of the vestibular nerve, which is instead pathognomonic of neurofibromatosis type 2 (NF2). Recently, a schwannomatosis family with a germline mutation of the SMARCB1 gene on chromosome 22 has been described. We report on the molecular analysis of the SMARCB1 and NF2 genes in a series of 21 patients with schwannomatosis and in eight schwannomatosis-associated tumors from four different patients. A novel germline SMARCB1 mutation was found in one patient; inactivating somatic mutations of NF2, associated with loss of heterozygosity (LOH) of 22q, were found in two schwannomas of this patient. This is the second report of a germline SMARCB1 mutation in patients affected by schwannomatosis and the first report of SMARCB1 mutations associated with somatic NF2 mutations in schwannomatosis-associated tumors. The latter observation suggests that a four-hit mechanism involving the SMARCB1 and NF2 genes may be implicated in schwannomatosis-related tumorigenesis.     

Type 1 papillary renal cell carcinoma in a patient with schwannomatosis: Mosaic versus loss of SMARCB1 expression in respectively schwannoma and renal tumor cells

In schwannomatosis, germline SMARCB1 or LZTR1 mutations predispose to the development of multiple benign schwannomas. Besides these, other tumors may occur in schwannomatosis patients. We present a 45‐year‐old male patient who developed multiple schwannomas and in addition a malignant type 1 papillary renal cell carcinoma (pRCC1). We identified a duplication of exon 7 of SMARCB1 on chromosome 22 in the constitutional DNA of the patient (c.796‐2246_986 + 5250dup7686), resulting in the generation of a premature stop codon in the second exon 7 copy (p.Glu330*). The mutant SMARCB1 allele proved to be retained in three schwannomas and in the pRCC1 of the patient. Loss of heterozygosity analysis demonstrated partial loss of the wild‐type SMARCB1 allele containing chromosome 22, suggesting loss of that chromosome in only a subset of tumor cells, in all four tumors. Immunohistochemical staining with a SMARCB1 antibody revealed a mosaic SMARCB1 expression pattern in the three benign schwannomas, but absence of expression in the malignant tumor cells of the pRCC1. To our knowledge, this difference in SMARCB1 protein expression has not been reported before. We conclude that a germline SMARCB1 mutation may predispose to the development of pRCC1, thereby further widening the spectrum of tumors that can develop in the context of schwannomatosis. © 2016 Wiley Periodicals, Inc.     

Update from the 2011 International Schwannomatosis Workshop: From genetics to diagnostic criteria

Schwannomatosis is the third major form of neurofibromatosis and is characterized by the development of multiple schwannomas in the absence of bilateral vestibular schwannomas. The 2011 Schwannomatosis Update was organized by the Children's Tumor Foundation ( www.ctf.org ) and held in Los Angeles, CA, from June 5–8, 2011. This article summarizes the highlights presented at the Conference and represents the “state‐of‐the‐field” in 2011. Genetic studies indicate that constitutional mutations in the SMARCB1 tumor suppressor gene occur in 40–50% of familial cases and in 8–10% of sporadic cases of schwannomatosis. Tumorigenesis is thought to occur through a four‐hit, three‐step model, beginning with a germline mutation in SMARCB1 (hit 1), followed by loss of a portion of chromosome 22 that contains the second SMARCB1 allele and one NF2 allele (hits 2 and 3), followed by mutation of the remaining wild‐type NF2 allele (hit 4). Insights from research on HIV and pediatric rhabdoid tumors have shed light on potential molecular pathways that are dysregulated in schwannomatosis‐related schwannomas. Mouse models of schwannomatosis have been developed and promise to further expand our understanding of tumorigenesis and the tumor microenvironment. Clinical reports have described the occurrence of intracranial meningiomas in schwannomatosis patients and in families with germline SMARCB1 mutations. The authors propose updated diagnostic criteria to incorporate new clinical and genetic findings since 2005. In the next 5 years, the authors expect that advances in basic research in the pathogenesis of schwannomatosis will lead toward clinical investigations of potential drug therapies. © 2013 Wiley Periodicals, Inc.     

Alterations in the SMARCB1 (INI1) tumor suppressor gene in familial schwannomatosis

Schwannomatosis is a third major form of neurofibromatosis that has recently been linked to mutations in the SMARCB1 (hSnf5/INI1) tumor suppressor gene. We analyzed the coding region of SMARCB1 by direct sequencing and multiplex ligation-dependent probe amplification (MLPA) in genomic DNA from 19 schwannomatosis kindreds. Microsatellite markers in the SMARCB1 region were developed to determine loss of heterozygosity (LOH) in associated tumors. We detected four alterations in conserved splice acceptor or donor sequences of exons 3, 4 and 6. Two alterations that likely affect splicing were seen in introns 4 and 5. An additional four alterations of unclear pathogenicity were found to segregate on the affected allele in eight families including two non-conservative missense alterations in three families. No constitutional deletions or duplications were detected by MLPA. Nine of 13 tumors examined showed partial LOH of the SMARCB1 region consistent with 'second hits.' Alterations were detected in tumors both with and without somatic NF2 gene changes. These findings support the hypothesis that SMARCB1 is a tumor suppressor for schwannomas in the context of familial disease. Further work is needed to determine its role in other multiple and single tumor syndromes.     

Constitutional LZTR1 mutation presenting with a unilateral vestibular schwannoma in a teenager

Schwannomatosis is a rare neurofibromatosis clinically diagnosed by age‐dependent criteria, with bilateral vestibular schwannoma and/or a constitutional NF2 mutation representing exclusion criteria. Following SMARCB1 germline mutations, constitutional mutations in LZTR1 were discovered. We report on the molecular investigation in a patient presenting at 14 years with a unilateral vestibular schwannoma, ultimately causing blindness and unilateral hearing loss, in the absence of other schwannomas or a positive family history. In DNA derived from frozen tumor tissue, a comprehensive NF2, SMARCB1 and LZTR1 analysis showed an NF2 truncating mutation c.1006_1021delins16; an LZTR1 mutation c.791+1G>A; and a partial 22q deletion including NF2, SMARCB1 and LZTR1. Sequence analysis on peripheral blood derived DNA showed the LZTR1 mutation to be constitutional, but the NF2 mutation and partial 22q deletion were not found, indicating them to be somatic events. RNA‐based targeted analysis confirmed missplicing of LZTR1 intron 8, predicted to result in a premature stop codon. This LZTR1 mutation was paternally inherited. While isolated vestibular schwannoma or NF2 may be considered in a young individual with a unilateral vestibular schwannoma, this report suggests that LZTR1 ‐related schwannomatosis be added to this differential diagnosis.     

A germline missense mutation in COQ6 is associated with susceptibility to familial schwannomatosis

PurposeSchwannomatosis, a subtype of neurofibromatosis, is characterized by multiple benign, nonvestibular, nonintradermal schwannomas. Although the tumor suppressor SMARCB1 gene has been frequently identified as the underlying genetic cause of half of familial and ~10% of sporadic schwannomatosis, for most other cases, further causative genes remain to be discovered. Herein, we characterize the genome of a schwannomatosis family without constitutional inactivation of the SMARCB1 gene to explore novel genomic alterations predisposing individuals to the familial disease.MethodsWe performed whole-genome/exome sequencing on genomic DNA of both schwannomatosis-affected and normal members of the family.ResultsWe identified a novel missense mutation (p.Asp208His; c.622G>C) in the coenzyme Q10 (CoQ10) biosynthesis monooxygenase 6 gene (COQ6) in schwannomatosis-affected members. The deleterious effects of the COQ6 mutations were validated by their lack of complementation in a coq6-deficient yeast mutant. Our study further indicated that the resultant haploinsufficiency of COQ6 might lead to CoQ10 deficiency and chronic overproduction of reactive oxygen species in Schwann cells.ConclusionAlthough the exact oncogenetic mechanisms in this schwannomatosis family remain to be elucidated, our data strongly indicate a probable role of COQ6 mutation and CoQ10 deficiency in the development of familial schwannomatosis.     

Novel germ-line deletion of SNF5/INI1/SMARCB1 gene in neonate presenting with congenital malignant rhabdoid tumor of kidney and brain primitive neuroectodermal tumor

We describe a neonate who had a rare tumor combination of a malignant rhabdoid tumor of the kidney (MRTK) and a brain primitive neuroectodermal tumor (PNET). Genetic alterations of the SNF5/INI1/SMARCB1 gene were investigated by PCR-single-strand conformation polymorphism (SSCP), loss of heterozygosity (LOH), sequence, and karyotyping analyses, and the gene expression level was determined by real-time quantitative RT-PCR analysis. PCR band signals of each exon of the hSNF5/INI1 were weak or nearly undetectable in both MRTK and PNET, whereas those of the corresponding normal kidney were clearly detected. Aberrantly migrating SSCP bands led to identification of a nucleotide change in intron 8. Although this was regarded as a polymorphism, only the changed nucleotide was observed in the normal kidney of the patient. Allelic states in the parents were heterozygous for the polymorphism in the father and homozygous for the normal sequence in the mother. Thus, it was evident that a substantial genetic part of the maternal normal allele including SNF5/INI1 was deleted as a de novo germ-line mutation. In both tumors, LOH at microsatellite loci on the long arm of chromosome 22 was evident, and expression of SNF5/INI1 mRNA was drastically decreased compared to that in control tissues (0.7-3.9 vs. 123.6-153.5). Deletion of a substantial genetic part demonstrated in our patient is the novel appearance of a germ-line deletion of the SNF5/INI1 gene. Additional large somatic deletions resulted in total inactivation of the gene in both tumors. Our patient provides evidence for an important role of SNF5/INI1germ-line mutation in predisposing patients to multiple rhabdoid tumors.     

Female germline mosaicism in tuberous sclerosis confirmed by molecular genetic analysis

We have investigated a family in which three siblings with the autosomal dominant disorder tuberous sclerosis had unaffected parents. The family were typed for polymorphic markers spanning the two genes known to cause tuberous sclerosis located at 9q34 (TSC1) and 16p13.3 (TSC2). TSC1 markers showed different maternal and paternal haplotypes in affected children, excluding a mutation in TSC1 as the cause of the disease. For the TSC2 markers all the affected children had the same maternal and paternal haplotypes, as did three of their unaffected siblings. Mutation screening by RT-PCR and direct sequencing of the TSC2 gene identified a 4 bp insertion TACT following nucleotide 2077 in exon 18 which was present in the three affected children but not in five unaffected siblings or the parents. This mutation would cause a frameshift and premature termination at codon 703. Absence of the mutation in lymphocyte DNA from the parents was consistent with germline mosaicism and this was confirmed by our finding of identical chromosome 16 haplotypes in affected and unaffected siblings, providing unequivocal evidence of two different cell lines in the gametes. Molecular analysis of the TSC2 alleles present in the affected subjects showed that the mutation had been inherited from the mother. This is the first case of germline mosaicism in tuberous sclerosis proven by molecular genetic analysis and also the first example of female germline mosaicism for a characterized autosomal dominant gene mutation apparently not associated with somatic mosaicism.      

Molecular confirmation of germ line mosaicism for a submicroscopic deletion of chromosome 22q11

Submicroscopic deletions of chromosome 22q11 have been reported in a multiple anomaly syndrome variously labelled as velocardiofacial syndrome, conotruncal anomaly face syndrome, and Di George syndrome. Most 22q11 microdeletions occur sporadically, although in some cases the deletion may be transmitted. We describe two affected sibs with confirmed 22q11 deletions from unaffected parents who are not deleted. Haplotype analysis demonstrates that the deletion in the affected sibs has occurred on the same maternal chromosome 22. Furthermore, an unaffected sib was found to have inherited the same maternal haplotype at 22q11 in an undeleted form. This is the first molecular demonstration of germ line mosaicism for a microdeletion at chromosome 22q11 and highlights the need for caution in estimation of recurrence risks, even when constitutional deletions have been excluded on parental analysis. Am. J. Med. Genet. 78:103–106, 1998. © 1998 Wiley-Liss, Inc.     

SWI/SNF deficient central nervous system neoplasms

The SWItch/Sucrose Non-Fermentable (SWI/SNF) complexes are ubiquitous ATP dependent chromatin remodeling complexes that provide epigenetic regulation of gene expressions across the genome. Different combination of SWI/SNF subunits allow tissue specific regulation of critical cellular processes. The identification of SMARCB1 inactivation in pediatric malignant rhabdoid tumors provided the first example that the SWI/SNF complex may act as a tumor suppressor. It is now estimated at least 20% of all human tumors contain mutations in the subunits of the SWI/SNF complex. This review summarizes the central nervous system tumors with alterations in the SWI/SNF complex genes. Atypical teratoid/rabdoid tumor (AT/RT) is a highly aggressive embryonal tumor genetically characterized by bi-allelic inactivation of SMARCB1, and immunohistochemically shows complete absence of nuclear expression of its protein product INI1. A small subset of AT/RT show retained INI1 expression but defects in another SWI/SNF complex gene SMARCA4. Embryonal tumors with medulloblastoma, pineoblastoma, or primitive neuroectodermal morphology but loss of INI1 expression are now classified as AT/RT. Cribriform neuroepithelial tumor (CRINET) is an intra or para-ventricular tumor that has similar SMARCB1 alterations as AT/RT but generally has a benign clinical course. Besides AT/RT and CRINET, compete loss of nuclear INI1 expression has also been reported in poorly differentiated chordoma and intracranial myxoid sarcoma within the central nervous system. Families with non-truncating SMARCB1 mutations are prone to develop schwannomatosis and a range of developmental syndromes. The schwannomas in these patients usually demonstrate a mosaic INI1 staining pattern suggestive of partial residual protein function. Finally, clear cell meningioma is a WHO grade II variant meningioma characterized by bi-allelic inactivation of the SMARCE1 gene and immunohistochemically show loss of its protein product BAF57 expression in tumor cell nuclei.     

Schwannomatosis presenting as pancreatic and submandibular gland schwannoma

Schwannomatosis is a well-established third form of neurofibromatosis, characterized by the presence of multiple non-vestibular, non-intradermal schwannomas, often associated with chronic pain. Herein, we report a 41-year-old man with a history of paternal neurofibromatosis 1, who presented with partially cystic tumors in the pancreas and in the right submandibular gland. Besides, he complained of neuropathic pain in the right inguinal and suprapubic area. Magnetic resonance imaging revealed multiple intradural-extramedullary tumors at the cervical, thoracic and lumbar spinal canal, suggestive of schwannomas. The vestibular nerves were not involved. Pathological examination of the glandular tumors disclosed benign schwannomas. These tumors had substantial myxoid stroma and prominent cystic change, and showed a mosaic pattern of loss of INI1/SMARCB1 expression by immunohistochemistry. Later, the patient developed three nodules in the right lung which were interpreted as schwannomas. To our knowledge, this is the first report of schwannomatosis presenting as pancreatic and salivary gland schwannomas.     

Expanding the mutational spectrum of LZTR1 in schwannomatosis

Schwannomatosis is characterized by the development of multiple non-vestibular, non-intradermal schwannomas. Constitutional inactivating variants in two genes, SMARCB1 and, very recently, LZTR1, have been reported. We performed exome sequencing of 13 schwannomatosis patients from 11 families without SMARCB1 deleterious variants. We identified four individuals with heterozygous loss-of-function variants in LZTR1. Sequencing of the germline of 60 additional patients identified 18 additional heterozygous variants in LZTR1. We identified LZTR1 variants in 43% and 30% of familial (three of the seven families) and sporadic patients, respectively. In addition, we tested LZTR1 protein immunostaining in 22 tumors from nine unrelated patients with and without LZTR1 deleterious variants. Tumors from individuals with LZTR1 variants lost the protein expression in at least a subset of tumor cells, consistent with a tumor suppressor mechanism. In conclusion, our study demonstrates that molecular analysis of LZTR1 may contribute to the molecular characterization of schwannomatosis patients, in addition to NF2 mutational analysis and the detection of chromosome 22 losses in tumor tissue. It will be especially useful in differentiating schwannomatosis from mosaic Neurofibromatosis type 2 (NF2). However, the role of LZTR1 in the pathogenesis of schwannomatosis needs further elucidation.     

Familial neonatal Marfan syndrome due to parental mosaicism of a missense mutation in the FBN1 gene

We present a family in which three siblings were born with neonatal Marfan syndrome (MFS) to unaffected parents. The clinical findings included joint contractures, large ears, loose skin, ectopia lentis, muscular hypoplasia, aortic root dilatation, mitral and tricuspid valve insufficiency, and pulmonary emphysema. All three siblings died due to cardiorespiratory insufficiency by 2-4 months of age. Screening of the FBN1 gene showed the heterozygous c.3257G > A (p.Cys1086Tyr) mutation in the proband. Mosaicism of the mutation was demonstrated in the somatic cells and in the germ line of the father. Although three examples of parental mosaicism for classical MFS were demonstrated previously, this is the first report of familial occurrence of neonatal MFS due to a heterozygous mutation in FBN1. In conclusion, the p.Cys1086Tyr mutation in FBN1 is consistently associated with neonatal MFS. Parental mosaicism should always be kept in mind when counseling families with MFS.     

SMARCB1/INI1 missense mutation in mucinous carcinoma with rhabdoid features

Malignant rhabdoid tumor (MRT) is a rare and aggressive tumor associated with deletion or mutation of a tumor suppressor gene SMARCB1/INI1, a member of the SWI/SNF chromatin-remodeling complex. Reported herein is a case of pancreatic mucinous carcinoma accompanying rhabdoid features with immunohistochemical and ultrastructural studies as well as analysis of the SMARCB1/INI1 gene. A 65-year-old woman presented with a 2 month history of abdominal and chest pain. A well-defined grayish tan fish-flesh mass (11 x 9 x 7 cm) with focal mucinous area was present in the pancreatic tail. Microscopically, the tumor had a biphasic growth pattern: a mucinous carcinoma component and a poorly differentiated carcinoma component with rhabdoid features showing loosely cohesive cells with abundant eosinophilic cytoplasm, displaced nuclei, and prominent nucleoli. The rhabdoid component coexpressed vimentin and cytokeratin. Sequencing analysis of the DNA extracted from the mucinous and rhabdoid components showed a missense mutation CCC to ACC in codon 116 of the SMARCB1/INI1 gene. Being aware of rhabdoid features would help diagnose this rare and aggressive malignant tumor and may provide an opportunity for further evaluation of SMARCB1/INI1 gene alteration and determination of its prognostic significance.     

Molecular study of frequency of mosaicism in neurofibromatosis 2 patients with bilateral vestibular schwannomas

Neurofibromatosis 2 (NF2) is a severe autosomal dominant disorder that predisposes to multiple tumours of the nervous system. About half of all patients are founders with clinically unaffected parents. The purpose of the present study was to examine the extent to which mosaicism is present in NF2 founders. A total of 233 NF2 founders with bilateral vestibular schwannomas (BVS) were screened by exon scanning. NF2 mutations were detected in the blood samples of 122 patients (52%). In 10 of the 122 cases, the ratio of mutant to normal alleles was obviously less than 1, suggesting mosaicism. Tumour specimens were available from 35 of the 111 subjects in whom no mutation could be detected in blood specimens. Mutational analysis by exon scanning detected typical NF2 mutations in 21 of the 35 tumours. In nine subjects, the alterations found in tumours could be confirmed to be the constitutional mutation based on finding of identical mutations in pathologically and/or anatomically distinct second tumours. In six other subjects with only a single tumour available, allelic loss of the NF2 gene was found in addition to the mutation in each tumour, suggesting that either the mutation or the deletion of the NF2 gene is probably the constitutional genetic alteration. Our results suggest that failure to find constitutional mutations in blood specimen from these 15 patients was not because of the limitation of the applied screening technique, but the lack of the mutations in their leucocytes, best explained by mosaicism. Extrapolating the rate (15/35 = 43%) of mosaicism in these 35 cases to the 111 NF2 founders with no constitutional NF2 mutations found in their blood, we inferred 48 mosaic subjects (111 x 0.429). Adding the 10 mosaic cases detected directly in blood specimens, we estimate the rate of mosaicism to be 24.8% (58/233) in our cohort of 233 NF2 founders with bilateral vestibular schwannomas.     

Infrequent SMARCB1/INI1 gene alteration in epithelioid sarcoma: a useful tool in distinguishing epithelioid sarcoma from malignant rhabdoid tumor

Loss of SMARCB1/INI1 protein expression is considered useful for confirming a histologic diagnosis of malignant rhabdoid tumor. However, loss of SMARCB1/INI1 protein expression has recently been reported in other tumors as well, including a few cases of epithelioid sarcoma. In addition, the histopathologic differences between proximal-type epithelioid sarcoma and malignant rhabdoid tumor have not been conclusively defined. We analyzed SMARCB1/INI1 protein expression in 54 epithelioid sarcoma (proximal-type, 25; distal-type, 29) and examined alterations of the SMARCB1/INI1 gene in the cases lacking protein expression. We found that 19 (76.0%) proximal-type epithelioid sarcoma and 27 (93.1%) distal-type epithelioid sarcoma showed loss of SMARCB1/INI1 protein expression. Analysis of 39 cases with loss of protein expression revealed 4 cases (10.3%) with SMARCB1/INI1 gene alterations at the DNA level (homozygous deletion, 2; 1- or 2-bp deletion, 2) that could have induced the loss of gene products, and all 4 of these were proximal-type epithelioid sarcoma. Epithelioid sarcoma was thus associated with a high frequency of loss of SMARCB1/INI1 protein expression similar to that in malignant rhabdoid tumor. However, the frequency of SMARCB1/INI1 gene alteration at the DNA level in proximal-type epithelioid sarcoma was significantly lower than that in malignant rhabdoid tumor. In addition, the prognosis of patients with malignant rhabdoid tumor is significantly worse than that of patients with proximal-type epithelioid sarcoma (P = .001). Therefore, proximal-type epithelioid sarcoma and malignant rhabdoid tumor are suggested to be distinctive tumors with respect to the mechanism of the loss of SMARCB1/INI1 protein expression. Analysis of alterations in the SMARCB1/INI1 gene may thus be a useful diagnostic tool to distinguish proximal-type epithelioid sarcoma from malignant rhabdoid tumor.     

The presence of germ line mosaicism in cleidocranial dysplasia

Cleidocranial dysplasia (CCD) is typically an autosomal dominant condition. The possibility of alternative causes, such as an autosomal recessive form or germ line mosaicism, have been suggested in some families with CCD, but not proven. We present a family consisting of a mother having three sons affected with CCD. One of the affected boys is a half brother to the other two affected children. The diagnosis of CCD was confirmed by DNA analysis of the RUNX2 gene in all three of the boys in blood; however, initial DNA testing in the mother's blood did not detect the presence of a RUNX2 mutation in the mother. Further testing through heteroduplex analysis applying high-resolution melting analysis followed by subcloning detected low-level mosaicism in DNA isolated from maternal blood and buccal swab, confirming low-level mosaicism in somatic cells. We present the first case of confirmed germ line mosaicism in CCD.