SMARCB1/INI1 germline mutations contribute to 10% of sporadic schwannomatosis

Background  

Schwannomatosis is a disease characterized by multiple non-vestibular schwannomas. Although biallelic NF2 mutations are found in schwannomas, no germ line event is detected in schwannomatosis patients. In contrast, germline mutations of the SMARCB1 (INI1) tumor suppressor gene were described in familial and sporadic schwannomatosis patients.     

High-resolution array-CGH profiling of germline and tumor-specific copy number alterations on chromosome 22 in patients affected with schwannomas

Schwannomas may develop sporadically or in association with NF2 and schwannomatosis. The fundamental aberration in schwannomas is the bi-allelic inactivation of the NF2 gene. However, clinical and molecular data suggest that these tumors share a common pathogenetic mechanism related to as yet undefined 22q-loci. Linkage studies in schwannomatosis, a condition related to NF2, have defined a candidate 22q-locus and excluded the NF2 gene as the causative germline mutation. Thus, analysis of aberrations in schwannomas may lead to the identification of putative gene(s) involved in the development of schwannoma/schwannomatosis. We profiled a series of 88 schwannomas and constitutional DNA using a tiling path chromosome 22 array. Array-CGH is a suitable method for high-resolution discrimination between germline and tumor-specific aberrations. Previously reported frequencies of 22q-associated deletions in schwannomas display large discrepancies, ranging from 30% to 80%. We detected heterozygous deletions in 53% of schwannomas and the predominant pattern was monosomy 22. In addition, three tumors displayed terminal deletions and four harbored overlapping interstitial deletions of various sizes encompassing the NF2 gene. When profiling constitutional DNA, we identified eight loci that were affected by copy number variation (CNV). Some of the identified CNVs may not be phenotypically neutral and the possible role of these CNVs in the pathogenesis of schwannomas should be studied further. We observed a correlation between the breakpoint position, present in tumor and/or constitutional DNA and the location of segmental duplications. This association implicates these unstable regions in rearrangements occurring both in meiosis and mitosis.     

Molecular Analysis of the NF2 Tumor-Suppressor Gene in Schwannomatosis

Patients with multiple schwannomas without vestibular schwannomas have been postulated to compose a distinct subclass of neurofibromatosis (NF), termed "schwannomatosis." To compare the molecular-genetic basis of schwannomatosis with NF2, we examined the NF2 locus in 20 unrelated schwannomatosis patients and their affected relatives. Tumors from these patients frequently harbored typical truncating mutations of the NF2 gene and loss of heterozygosity of the surrounding region of chromosome 22. Surprisingly, unlike patients with NF2, no heterozygous NF2-gene changes were seen in normal tissues. Examination of multiple tumors from the same patient revealed that some schwannomatosis patients are somatic mosaics for NF2-gene changes. By contrast, other individuals, particularly those with a positive family history, appear to have an inherited predisposition to formation of tumors that carry somatic alterations of the NF2 gene. Further work is needed to define the pathogenetics of this unusual disease mechanism.     

Incidence and classification of pediatric diffuse parenchymal lung diseases in Germany

Neurofibromatosis type 2 (NF2) is a tumour-prone disorder characterised by the development of multiple schwannomas and meningiomas. Prevalence (initially estimated at 1: 200,000) is around 1 in 60,000. Affected individuals inevitably develop schwannomas, typically affecting both vestibular nerves and leading to hearing loss and deafness. The majority of patients present with hearing loss, which is usually unilateral at onset and may be accompanied or preceded by tinnitus. Vestibular schwannomas may also cause dizziness or imbalance as a first symptom. Nausea, vomiting or true vertigo are rare symptoms, except in late-stage disease. The other main tumours are schwannomas of the other cranial, spinal and peripheral nerves; meningiomas both intracranial (including optic nerve meningiomas) and intraspinal, and some low-grade central nervous system malignancies (ependymomas). Ophthalmic features are also prominent and include reduced visual acuity and cataract. About 70% of NF2 patients have skin tumours (intracutaneous plaque-like lesions or more deep-seated subcutaneous nodular tumours). Neurofibromatosis type 2 is a dominantly inherited tumour predisposition syndrome caused by mutations in the NF2 gene on chromosome 22. More than 50% of patients represent new mutations and as many as one-third are mosaic for the underlying disease-causing mutation. Although truncating mutations (nonsense and frameshifts) are the most frequent germline event and cause the most severe disease, single and multiple exon deletions are common. A strategy for detection of the latter is vital for a sensitive analysis. Diagnosis is based on clinical and neuroimaging studies. Presymptomatic genetic testing is an integral part of the management of NF2 families. Prenatal diagnosis and pre-implantation genetic diagnosis is possible. The main differential diagnosis of NF2 is schwannomatosis. NF2 represents a difficult management problem with most patients facing substantial morbidity and reduced life expectancy. Surgery remains the focus of current management although watchful waiting with careful surveillance and occasionally radiation treatment have a role. Prognosis is adversely affected by early age at onset, a higher number of meningiomas and having a truncating mutation. In the future, the development of tailored drug therapies aimed at the genetic level are likely to provide huge improvements for this devastating condition.     

Identification of three neurofibromatosis type 2 (NF2) gene mutations in vestibular schwannomas

Vestibular schwannomas (VSs) are common benign tumors of Schwann cell origin and are frequently found in patients with neurofibromatosis type 2 (NF2). We analyzed 15 sporadic VSs for mutations in the NF2 gene. We detected mutations in three of the tumors, two of which contained loss of heterozygosity (LOH). One of the tumors contained a novel mutation, a 19-bp deletion in exon 4. The two other tumors contained an identical mutation, a complete exon 4 deletion. The exon 4 deletion represents the second most frequently reported mutation of the NF2 gene in VSs.     

The mouse ortholog of the human SMARCB1 gene encodes two splice forms

The human SMARCB1 gene (SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily b, member 1, previously named the INI1/hSNF5 gene) is a tumor suppressor gene located on chromosome 22q11.2 and is inactivated in malignant rhabdoid tumors. By using an EST-based approach, we cloned two splice forms of the Smarcb1 gene in mouse and a longer splice form of the human ortholog. Proteins corresponding to the longer (385 aa) and the shorter (376 aa) forms are 100% conserved between human and mouse. Meningiomas and schwannomas are tumors frequently deleting various regions on chromosome 22, including the SMARCB1 locus. We therefore directly sequenced seven SMARCB1 exons (90% of the open reading frame) in search for mutations in 41 meningiomas and 23 schwannomas. No inactivating mutations were observed, which suggests that the SMARCB1 gene is not involved in the pathogenesis of these tumors.     

Constitutional Mutations of the hSNF5/INI1 Gene Predispose to a Variety of Cancers

Biallelic, truncating mutations of the hSNF5/INI1 gene have recently been documented in malignant rhabdoid tumor (MRT), one of the most aggressive human cancers. This finding suggests that hSNF5/INI1 is a new tumor-suppressor gene for which germline mutations might predispose to cancer. We now report the presence of loss-of-function mutations of this gene in the constitutional DNA from affected members but not from healthy relatives in cancer-prone families. Furthermore, a constitutional mutation is documented in a patient with two successive primary cancers. In agreement with the two-hit model, the wild-type hSNF5/INI1 allele is deleted in the tumor DNA from mutation carriers. In all tested cases, DNA from parents demonstrated normal hSNF5/INI1 sequences, therefore indicating the de novo occurrence of the mutation, which was shown to involve the maternal allele in one case and the paternal allele in two other cases. These data indicate that constitutional mutation of the hSNF5/INI1 gene defines a new hereditary syndrome predisposing to renal or extrarenal MRT and to a variety of tumors of the CNS, including choroid plexus carcinoma, medulloblastoma, and central primitive neuroectodermal tumor. This condition, which we propose to term "rhabdoid predisposition syndrome," may account for previous observations of familial and multifocal cases of the aforementioned tumor types. It could also provide the molecular basis for cases of Li-Fraumeni syndrome without p53 germline mutations.     

HeLa细胞中INI1通过与E2A作用下调CD117

目的:已经证明,CD117基因转录启始位点的上游-480bp包括4个E-box和一个GC-box。试验要确定INI1是否通过与E-box结合蛋白的相互作用来调控CD117。方法与结果:实验证明INI1过表达下调 CD117的mRNA水平和荧光素酶的活性,这个酶通过CD117启动子来启动。通过染色质共免疫沉淀反应实验,发现在HeLa细胞未损伤的染色质里面,INI1可以特定的结合CD117的启动子。绘制INI1在CD117启动子区域的结合位点图表明, E-boxes而不是GC-box对于下调CD117启动子的活性是必须的,这一点在Co-IP(共免疫沉淀)实验中被进一步的证实,在这个试验中INI1与E-box结合蛋白E2A在体内相互作用。结论:总的来说,INI1参与调节CD117的表达和细胞增殖。     

INI1 expression induces cell cycle arrest and markers of senescence in malignant rhabdoid tumor cells

The INI1 gene, which encodes a functionally uncharacterized protein component of the hSWI/SNF chromatin remodeling complex, is often mutated or deleted in malignant rhabdoid tumor (MRT). Two isoforms of INI1, that differ by the variable inclusion of nine amino acids, potentially are produced by differential RNA splicing. To determine the effect of the two INI1 isoforms on cell growth, INI1-devoid (MRT) and INI1-expressing cell lines were transfected separately with mammalian expression vectors or transduced with adenoviruses. Transfection of the short form of INI1 into either INI1-deficient or expressing cell lines resulted in complete suppression of cell growth in colony formation assays. The longer splice variant induced moderate to severe growth suppression of MRT cells, but had a far milder effect on non-MRT cells. Transduction of MRT cells with adenoviruses expressing either isoform of INI1 led to a dramatic change in morphology, growth suppression, and cell cycle arrest. Furthermore, senescence-associated proteins were up-regulated after transduction, while levels of proteins implicated in cell cycle progression were down-regulated. Adenoviral delivery of INI1 into a non-MRT cell line, however, had no demonstrable effect on any of these parameters. These results support the genetic evidence that INI1 is a tumor suppressor gene gone awry in MRT cells, and also suggest that delivery of the INI1 gene to MRT cells by adenoviruses may lead to a more effective treatment of this highly aggressive malignancy.     

A group of schwannomas with interstitial deletions on 22q located outside the NF2 locus shows no detectable mutations in the NF2 gene

Schwannomas are tumors arising mainly at cranial and spinal nerves. Bilateral vestibular schwannoma is the hallmark of neurofibromatosis type 2 (NF2). The NF2 gene has been cloned and comprehensive analysis of its mutations in schwannomas shows that up to 60% of tumors carry inactivating mutations. Thus, the genetic mechanism behind the development of more than 40% of schwannomas without NF2 mutations is unknown. We have therefore studied tumor tissue from 50 human schwannomas by allelotyping and have found chromosome 22 deletions in over 80% of the cases. We detected 14 cases (27%) that revealed partial deletions of one copy of chromosome 22, i.e., terminal and/or interstitial deletions. We sequenced the NF2 gene in seven of these tumors and detected only one case with mutations. The deletion mapping of chromosome 22 in tumors with partial deletions indicates that several regions, in addition to the NF2 locus, harbor genes involved in schwannoma tumorigenesis. Our findings suggest that heterogeneity in the mechanisms leading to the development of schwannomas probably exists. These findings are in agreement with the recent analysis of schwannomas from familial and sporadic cases of schwannomatosis and point to a possible role of an additional gene, which, in cooperation with the NF2 tumor suppressor, causes schwannomas. Received: 12 November 1998 / Accepted: 1 March 1999     

人肝细胞癌中抑癌基因PTEN/MMAC1/TEP1的突变分析

PTEN／MMAC1／TEP1(PTEN)是新近分离到的抑癌基因,在多种肿瘤中存在突变。我们检测了34例人肝细胞癌中PTEN基因第5外显子和第8外显子的突变。采用聚合酶链方法以内含子引物扩增第5和第8外显子,继之以单链构象多态性和测序技术分析PTEN基因突变。有4例肝细胞癌SSCP显示异常条带并经测序证实存在突变。2例发生于第4内含子,突变位点相同;另两例发生于第8外显子,其中1例碱基颠换导致PTEN蛋白产物304位半胱氨酸突变为甘氨酸。     

人肝细胞癌中抑癌基因PTEN／MMAC1／TEP1的突变分析

PTEN/MMAC1/TEP1 is a tumor suppressor gene. Its mutation has been found in several different types of human cancers. 34 primary human hepatocellular carcinomas have been examined for mutations in exon 5 and exon 8 of the PTEN gene. Exon 5 and exon 8 were amplified by polymerase chain reaction (PCR) with intronic primers and subjected to single strand conformation polymorphism (SSCP) analysis. SSCPs were found in 4 of the 34 hepatocellular carcinomas analyzed. Direct sequencing of the PCR products identified single base-pair substitutions in the four tumor DNA samples, two in intron 4 and two in exon 8. One of the base-pair substitution in exon 8 is a missense mutation, which changed codon 304 of PTEN protein from Cys to Gly. These data suggest that PTEN may be involved in the carcinogenesis and development of hepatocellular carcinoma.     

A novel mutation in the neurofibromatosis type 1 (NF1) gene promotes skipping of two exons by preventing exon definition

Using a protein truncation assay, we have identified a new mutation in the neurofibromatosis type 1 (NF1) gene that causes a severe defect in NF1 pre-mRNA splicing. The mutation, which consists of a G to A transition at position +1 of the 5' splice site of exon 12a, is associated with the loss of both exons 11 and 12a in the NF1 mRNA. Through the use of in vivo and in vitro splicing assays, we show that the mutation inactivates the 5' splice site of exon 12a, and prevents the definition of exon 12a, a process that is normally required to stimulate the weak 3' splice site of exon 12a. Because the 5' splice site mutation weakens the interaction of splicing factors with the 3' splice site of exon 12a, we propose that exon 11/exon 12a splicing is also compromised, leading to the exclusion of both exons 11 and 12a. Our results provide in vivo support for the importance of the exon definition model during NF1 splicing, and suggest that the NF1 region containing exons 11 and 12a plays an important role in the activity of neurofibromin.     

一个遗传性胰腺炎家系中新发现的胰蛋白酶原基因突变

对1个遗传性胰腺炎(hereditary pancreatitis, HP)家系中6例成员和120例无亲缘关系健康人的胰蛋白酶原基因(protease serine 1, PRSS1)进行PCR扩增, 产物纯化后测序, 结合受检者的血清肿瘤标志物、糖尿病相关生化指标以及近亲属的一般临床资料进行分析。结果发现4例家系成员PRSS1基因3号外显子区136位碱基存在C→T杂合性突变, 他们的基因型表现为野生型与突变型杂合现象, 另外在先证者PRSS1基因的3号外显子区171位碱基还存在着一个同义突变点(C→T), 而对照组和家系其他成员中未发现此两种突变, 突变阳性患者表现为乳酸、糖基化血红蛋白和糖类肿瘤标志物(CA19-9、CA125)增高。因此, PRSS1基因3号外显子区136位碱基C→T杂合性突变与该家系遗传性胰腺炎有关, 是该家系中遗传性胰腺炎的遗传易感因素。     

Neurofibromatosis type 2 attributable to gonosomal mosaicism in a clinically normal mother, and identification of seven novel mutations in the NF2 gene

Neurofibromatosis type 2 (NF2) is an autosomal dominant cancer syndrome that predisposes to the development of bilateral vestibular schwannomas sometimes associated with schwannomas at other locations, meningiomas, ependymomas and juvenile posterior subcapsular lenticular opacities. This disease is caused by inactivating mutations in the NF2 tumour-suppressor gene, located in 22q12. Recently, somatic mosaicism has been demonstrated in some "de novo" NF2 patients. We here report the genetic study of 33 NF2 patients from 33 unrelated Italian families. Twelve mutations were characterised, including seven newly identified mutations and five recurrent ones. Furthermore, we describe one patient with an inactivating mutation that lies in exon 13 but that is present in only a portion of the lymphocytes and, more importantly, a clinically normal individual carrying a somatic/germinal mosaicism for a nonsense mutation in exon 10 of the NF2 gene. Our results confirm the relatively high percentage of mosaicism for mutations in the NF2 gene and establish the importance of evaluating genomic DNA from several tissues, in addition to lymphocytes, so as to identify mosaicism in "de novo" NF2 patients and their relatives. In addition, the demonstration of somatic and/or gonadal mosaicism is an important tool for accurate genetic counselling in families with sporadic cases of NF2.     

Identification of a novel nonsynonymous mutation of EYA1 disrupting splice site in a Korean patient with BOR syndrome

The EYA1 gene is known as the causative gene of BOR (Branchio-oto-renal) syndrome which is a genetic disorder associated with branchial cleft cysts of fistulae, hearing loss, ear malformation, and renal anomalies. Although approximately 40 % of patients with BOR syndrome have mutations in the EYA1 gene and over 130 disease-causing mutations in EYA1 have been reported in various populations, only a few mutations have been reported in Korean families. In this study, genetic analysis of the EYA1 gene was performed in a Korean patient diagnosed with BOR syndrome and his parents. A de novo novel missense mutation, c.418G>A, located at the end of exon 6, changed glycine to serine at amino acid position 140 (p.G140S) and was suspected to affect normal splicing. Our in vitro splicing assay demonstrated that this mutation causes exon 6 skipping leading to frameshift and truncation of the protein to result in the loss of eyaHR. To the best of our knowledge, this is the first report revealing that a missense mutation in the exon disturbs normal splicing as a result of a substitution of the last nucleotide of an exon in EYA1.     

Expression of PiM-and PiZ-mutated forms of the human alpha 1-antitrypsin gene in transfected monkey COS1 cells

The PiZ mutation of the gene coding for alpha 1-antitrypsin results in a serum deficiency of this protein leading to early onset emphysema and liver disease. The PiZ gene has a Z-specific point mutation in exon V together with a point mutation in exon III which is also present in some normal (PiM) individuals. There has thus far been no system to study the effects of PiZ point mutations in tissue culture. We constructed plasmids containing alpha 1-antitrypsin cDNA synthetically altered at either exon III or exon V mutation sites and linked to simian virus 40 promoter sequences. Such constructs with the exon V mutation were transfected into monkey COS1 cells followed by analysis of expression of alpha 1-antitrypsin gene products. COS1 cells normally synthesize virtually no alpha 1-antitrypsin mRNA or protein. alpha 1-Antitrypsin mRNA is transcribed at high levels in cells transfected with either M or Z plasmids. Immunologic staining of COS1 cells within 48 h of transfection localizes alpha 1-antitrypsin protein to specific regions of the cytoplasm. This extranuclear localization is also observed with human HepG2 hepatoma cells, which synthesize alpha 1-antitrypsin at high levels, and with human SK-Hep1 hepatoma cells transfected with an M plasmid. The cloned synthetically altered alpha 1-antitrypsin genes provide a system for dissecting contributions of distinct point mutations to the pathological effects of the PiZ protein.