Germline SMARCB1 mutation and somatic NF2 mutations in familial multiple meningiomas.

BACKGROUND: Multiple meningiomas occur in <10% of meningioma patients. Their development may be caused by the presence of a predisposing germline mutation in the neurofibromatosis type 2 (NF2) gene. The predisposing gene in patients with non-NF2 associated multiple meningiomas remains to be identified. Recently, SMARCB1 was reported to be a potential predisposing gene for multiple meningiomas in a family with schwannomatosis and multiple meningiomas. However, involvement of this gene in the development of the meningiomas was not demonstrated. RESULTS: Five affected members of a large family with multiple meningiomas were investigated for the presence of mutations in SMARCB1 and NF2. A missense mutation was identified in exon 2 of SMARCB1 as the causative germline mutation predisposing to multiple meningiomas; furthermore, it was demonstrated that, in accordance with the two-hit hypothesis for tumourigenesis, the mutant allele was retained and the wild-type allele lost in all four investigated meningiomas. In addition, independent somatically acquired NF2 mutations were identified in two meningiomas of one patient with concomitant losses of the wild-type NF2 allele. CONCLUSION: It is concluded that, analogous to the genetic events in a subset of schwannomatosis associated schwannomas, a four-hit mechanism of tumour suppressor gene inactivation, involving SMARCB1 and NF2, might be operative in familial multiple meningiomas associated meningiomas.

SMARCB1/INI1 maternal germ line mosaicism in schwannomatosis.

Schwannomatosis is characterized by the development of multiple schwannomas of the nervous system, but without the occurrence of vestibular schwannomas. Most cases of schwannomatosis are thought to be sporadic, representing the first case in a family due to a new mutation in the causative gene. We recently identified SMARCB1/INI1 as a schwannomatosis-predisposing gene. Here, we analyzed this gene in a schwannomatosis family with two affected children, but with clinically unaffected parents. Both affected individuals carried a constitutional SMARCB1 mutation, c.1118+ 1G>A, that changes the donor splice site sequence of intron 8, causing skipping of exon 8 and resulting in the in-frame deletion of 132 nucleotides in the transcript. The mutation was not evident in constitutional DNA of the parents. Haplotyping revealed that the chromosome 22 segment that carries the mutant SMARCB1 allele originated from the mother. She transferred the same chromosome 22 segment, however, with a wild-type SMARCB1 copy, to a third unaffected child. Our findings indicate that the mother is germ line mosaic for the SMARCB1 mutation. In conclusion, our study shows for the first time that germ line mosaicism may occur in schwannomatosis, which has implications for genetic counseling in this disease.

Molecular characterization of areas with low grade tumor or satellitosis in human malignant astrocytomas.

Malignant astrocytomas often display histopathological heterogeneity. In the present study, we have molecularly characterized different areas within 4 such tumors to determine whether the tissue heterogeneity can be explained by differences in DNA constitution. Two tumors contained low grade areas, and the other 2 had areas with satellitosis. The tumors were examined for loss of heterozygosity with markers from chromosomes 9p, 10, and 17p and for amplification of the epidermal growth factor receptor gene. In each case, the high grade portion of the tumor displayed at least one of these structural alterations. However, identical alterations were found in the associated low grade or satellitosis areas of each tumor. Our data suggest that: (a) genetic alterations associated with tumor progression already occur in histopathologically low grade areas of high grade astrocytoma; (b) satellitosis associated with a high grade astrocytoma has to be considered as part of that tumor; and (c) tissue heterogeneity within a high grade astrocytoma is not a consequence of differences in DNA constitution at the loci that were examined.

The site of action of 2,4-dinitrophenol and salicylic acid upon the uncoupler-induced K+ efflux from non-metabolizing yeast.

Stimulation of K+ efflux from non-metabolizing yeast cells by 2,4-dinitrophenol or by salicylic acid occurs only after accumulation of the compounds into the cells, indicating that the site of action of the uncouplers is inside the cells. A correlation is found between the partition ratio of the lipophilic cation dibenzyldimethylammonium between cells and medium and the rate of K+ efflux.

Microsatellite instability and promoter methylation as possible causes of NF1 gene inactivation in neurofibromas.

Neurofibromatosis type 1 (NF1) is a frequent hereditary disorder. One of the characteristic features of this disease is the development of neurofibromas. Since the NF1 gene is supposed to be a tumour suppressor gene, these neurofibromas should develop upon inactivation of both NF1 alleles. So far, mutation and deletion have been found to be involved in NF1 gene inactivation. However, these inactivating mechanisms explain the development of only a limited fraction of analysed neurofibromas. In this study, we investigated microsatellite instability (MSI) and promoter methylation as potential contributors to NF1 gene inactivation. As site-specific methylation in the NF1 promoter inhibits binding of transcription factors Sp1 and CREB, we studied the methylation status of their binding sites in particular. We analysed 20 neurofibromas and three neurofibrosarcomas, but did not find evidence for microsatellite instability or NF1 promoter methylation in any of the tumours. Thus, our data suggest that both microsatellite instability and promoter methylation are unlikely to be the major causes of NF1 gene inactivation in these tumours.

Mechanism of spreading of the highly related neurofibromatosis type 1 (NF1) pseudogenes on chromosomes 2, 14 and 22.

Neurofibromatosis type 1 (NF1) is a frequent hereditary disorder that involves tissues derived from the embryonic neural crest. Besides the functional gene on chromosome arm 17q, NF1-related sequences (pseudogenes) are present on a number of chromosomes including 2, 12, 14, 15, 18, 21, and 22. We elucidated the complete nucleotide sequence of the NF1 pseudogene on chromosome 22. Only the middle part of the functional gene but not exons 21-27a, encoding the functionally important GAP-related domain of the NF1 protein, is presented in this pseudogene. In addition to the two known NF1 pseudogenes on chromosome 14 we identified two novel variants. A phylogenetic tree was constructed, from which we concluded that the NF1 pseudogenes on chromosomes 2, 14, and 22 are closely related to each other. Clones containing one of these pseudogenes cross-hybridised with the other pseudogenes in this subset, but did not reveal any in situ hybridisation with the functional NF1 gene or with NF1 pseudogenes on other chromosomes. This suggests that their hybridisation specificity is mainly determined by homologous sequences flanking the pseudogenes. Strong support for this concept was obtained by sequence analysis of the flanking regions, which revealed more than 95% homology. We hypothesise that during evolution this subset of NF1 pseudogenes initially arose by duplication and transposition of the middle part of the functional NF1 gene to chromosome 2. Subsequently, a much larger fragment, including flanking sequences, was duplicated and gave rise to the current NF1 pseudogene copies on chromosomes 14 and 22.

Nucleotide sequence of the filamentous bacteriophage M13 DNA genome: comparison with phage fd.

The 6407 nucleotide-long sequence of bacteriophage M13 DNA has been determined using both the chemical degradation and chain-termination methods of DNA sequencing. This sequence has been compared with that of the closely related bacteriophage fd (Beck et al., 1978). M13 DNA appears to be only a single nucleotide shorter than fd DNA. There is an average of 3.0% of nucleotide-sequence differences between the two genomes, but the distribution of these changes is not random; the sequence of some genes is more conserved than of others. In contrast, the nucleotide sequences and positions of the regulatory elements involved in transcription, translation and replication appear to be identical in both filamentous phage DNA genomes.

Glioneuronal tumors and medically intractable epilepsy: a clinical study with long-term follow-up of seizure outcome after surgery.

The present study intends to identify factors that predict postoperative clinical outcome in patients with gangliogliomas (GG) and dysembryoplastic neuroepithelial tumors (DNT). We evaluated the medical records of 45 patients with GG and 13 patients with DNT, treated surgically between 1985 and 1995. We assessed several clinical and histopathological features and analyzed the data statistically. At 5 years postoperatively, 63% of patients with GG and 58% of patients with DNT were seizure-free (Engel's class I). Younger age at surgery (P<0.01 for GG and P<0.05 for DNT), total resection (P<0.01 for GG), shorter duration of epilepsy (P<0.01), absence of generalized seizures (P<0.01 for GG; P<0.05 for DNT) and absence of epileptiform discharge in the post-operative EEG (P<0.01 for GG; P=0.01 for DNT) predicted a better postoperative seizure outcome. Tumor recurrence with malignant progression occurred in eight histologically benign GG and two anaplastic GG and was associated which older age at surgery (P=0.01) and subtotal resection of the tumor (P<0.01). Our results indicate that a prompt diagnosis, relatively soon after seizure onset, followed by complete resection of glioneuronal tumors provides the best chance for curing epilepsy and preventing their malignant transformation.

Polyposis coli, craniofacial exostosis and astrocytoma: the concomitant occurrence of the Gardner's and Turcot syndromes.

BACKGROUND: Up to 60% of the patients with known adenomatous polyposis coli may present hyperostosis of the skull and facial bones, and/or a susceptibility to fibromas. This is known as the Gardner's syndrome, and is considered as an allelic variant of familial adenomatous polyposis (FAP). Also, although very rare, an adenomatous polyposis coli may occur with malignant tumors of the central nervous system, known as Turcot syndrome. If both syndromes are different phenotypic presentation of FAP, this would explain a simultaneous occurrence. METHOD: We report the history of a patient who showed clinical signs of the simultaneous occurrence of both Gardner's and Turcot syndromes. The syndromes are compared, and in view of the literature, a genetic explanation for the concomitant occurrence is discussed. RESULTS: Evidence obtained from the literature to consider Turcot syndrome as a phenotype of FAB is as follows: (1) The occurrence of Gardner's and Turcot syndromes in one family, but in different members; (2) The presence of congenital hypertrophic retinal pigmented epithelium (CHRPE), which correlates with the expression of polyps in FAP patients, in both syndromes; (3) Linkage of the Turcot phenotype to the adenomatous polyposis coli locus by genetic markers. Evidence obtained from this case report indicates that there is a manifestation of both syndromes in one patient together with a positive family history for FAP. CONCLUSION: This concomitant occurrence of both Gardner's and Turcot syndromes in one patient clinically supports genetic and ophthalmic investigation to consider Turcot syndrome (like Gardner's syndrome) as a phenotypic variant of FAP. Patients with FAP should be examined for the presence of Gardner's syndrome. In case a Gardner's syndrome is suspected, a computed tomography scan of the brain is recommended because of the possible existence of a simultaneous Turcot syndrome.

[Molecular-genetic aspects of neurofibromatosis]. / Moleculair-genetische aspecten van neurofibromatosis.

Two forms of neurofibromatosis, type 1 (NF1) and type 2 (NF2) are connected with genes localized on chromosomes 17 and 22, respectively. The genes that are inactivated in neurofibromatosis code for the proteins neurofibromine and merline, respectively. Since inactivation leads to neoplasia, they are called tumour suppressor genes. Neurofibromine shows resemblances to proteins that serve to inactivate oncogenes. Merline has a relationship with proteins that connect the cytoskeleton and the cell membrane. The precise function of the proteins is still unknown. The NF1 gene is characterized by extraordinarily high sensitivity to mutation; half the NF1 patients have not inherited the disease. In the familial form of neurofibromatosis, a mutated gene is inherited and the normal allele in the tumour is inactivated, making tumour growth possible. In the sporadic form of neurofibromatosis, both normal alleles are inactivated locally in the tissue so that a tumour develops in that place.

Secondary meningioma in a long-term survivor of atypical teratoid/rhabdoid tumour with a germline INI1 mutation.

OBJECTIVE: We report on a patient who developed a meningioma more than two decades after removal at a young age of an atypical teratoid/rhabdoid tumour (AT/RT), which was due to a germline INI1 mutation, and radio- and chemotherapy. MATERIALS AND METHODS: We present genetic evidence that the meningioma is not a recurrence or metastasis of the AT/RT and not due to the INI1 mutation, but is a radiation-induced tumour. CONCLUSION: This is the first case illustrating that improved survival of young patients with an AT/RT after aggressive treatment may be gained at the cost of an increased risk for the development of radiation-induced, non-INI1-related tumours.

Long-term survival and transmission of INI1-mutation via nonpenetrant males in a family with rhabdoid tumour predisposition syndrome.

Rhabdoid tumour predisposition syndrome (RTPS) is a rare syndrome caused by inheritance of a mutated INI1 gene for which only two multigeneration families have been reported. To further characterise the genotype and phenotype of RTPS, we present a third family in which at least three cousins developed an atypical teratoid/rhabdoid tumour (AT/RT) at a young age. Two of these patients showed unusual long survival, and one of these developed an intracranial meningioma and a myoepithelioma of the lip in adulthood. Mutation analysis of INI1 revealed a germline G>A mutation in the donor splice site of exon 4 (c.500+1G>A) in the patients and in their unaffected fathers. This mutation prevents normal splicing and concomitantly generates a stop codon, resulting in nonsense-mediated mRNA decay. Biallelic inactivation of INI1 in the tumours, except for the meningioma, was confirmed by absence of nuclear INI1-protein staining. The myoepithelioma of one of the patients carried an identical somatic rearrangement in the NF2 gene as the AT/RT, indicating that both tumours originated from a common precursor cell. In conclusion, this study demonstrates for the first time transmission of a germline INI1-mutation in a RTPS family via nonpenetrant males, long-term survival of two members of this family with an AT/RT, and involvement of INI1 in the pathogenesis of myoepithelioma.

A region of common deletion in 22q13.3 in human glioma associated with astrocytoma progression.

Loss of heterozygosity for chromosome 22 (LOH 22) occurs in gliomas of all malignancy grades. Neurofibromatosis type 2 (NF2) patients are at increased risk of developing a glioma. However, the NF2 gene in 22q12.2 is not involved in glioma tumorigenesis. To detect additional regions on chromosome 22 that may harbor tumor suppressor genes important in glioma tumorigenesis, we determined LOH 22 profiles for 159 gliomas using 32 markers. LOH 22 was found in 46 tumors (29%). Thirteen tumors displayed partial LOH 22, from which we deduced a region of common deletion between markers D22S928 and D22S1169 in 22q13.3. LOH of at least this region was detected in 13% of the astrocytomas (As), in 20% of the anaplastic astrocytomas (AAs) and in 35% of the glioblastomas multiforme (GBMs). The significant increased frequency of LOH 22q13.3 in the highest malignancy grade (GBM vs. A and AA, p = 0.02) indicates that loss of this region is associated with astrocytoma progression.

Lampbrush loop-specific protein of Drosophila hydei.

By immunofluorescence techniques and protein blotting experiments we have shown that an antiserum specifically reacts with a Mr 80,000 protein (the "Ps protein") in the lampbrush loop " pseudonucleolus " in spermatocyte nuclei of Drosophila hydei. Comparative studies of X/Y and X/0 testes indicate that the gene encoding the Ps protein is not located on the Y chromosome but on an autosome or the X chromosome. The Ps protein is tissue specific. It is likely to be a rather conserved protein since the antigenic determinant recognized by the antiserum could be detected in the spermatocyte nuclei of a number of other Drosophila species. For those species with prominent Y chromosomal lampbrush loops, it could be shown that the cross-reaction is, as in D. hydei, associated with a specific Y chromosomal loop.

Molecular-genetic characterisation of gliomas that recur as same grade or higher grade tumours.

BACKGROUND: Due to their invasive growth, gliomas usually cannot be removed completely and almost always recur as same grade or higher grade malignancies. OBJECTIVE: To determine whether there were differences in the accumulation of genetic changes between the two types of glioma recurrence. METHODS: We genetically characterised 14 cases of lower grade glioma with a same grade recurrence, 12 cases of glioblastoma recurrence, and 14 cases of lower grade glioma with a higher grade recurrence. We investigated LOH (loss of heterozygosity) at 1p36, 10p15, the PTEN region in 10q23, the DMBT1 region in 10q25, 19q13, 22q13, LOH and mutation of TP53, and EGFR amplification. RESULTS: Genetic heterogeneity in the primary tumour was inferred in 3 cases of lower grade glioma with a higher grade recurrence. The cases of lower grade glioma with a higher grade recurrence displayed increased genetic instability in the recurrence (mean of 2.0 additional genetic changes per case) compared to cases with a same lower grade recurrence or those with a glioblastoma recurrence (mean of 0.6 and 0.8 additional changes per case, respectively). Compared to unselected primary glioblastomas, the glioblastomas that recurred as an operable tumour had infrequent EGFR amplification (8% v 30-40% of cases). CONCLUSIONS: Gliomas recurring as higher grade lesions might be genetically heterogeneous and accumulate more genetic changes than gliomas recurring as same grade lesions (whether originally low or high grade). Primary glioblastomas from patients for which the recurrence is operated because of prognostically more favourable clinical indices have infrequent EGFR amplification.

Involvement of Y chromosomal loci in the synthesis of Drosophila hydei sperm proteins.

A comparative study of the protein patterns in testes of wild-type and X/O flies of Drosophila hydei revealed quantitative differences in at least three major protein fractions. One protein component of a Mr 155,000 fraction and a protein of Mr 35,000 are completely absent in X/O testes. The amount of protein in a Mr 55,000 fraction is considerably reduced. The tubulins, which are part of this fraction, are also reduced in amount. All three proteins were found as constituents of sperm tails. Studies of Y chromosomal mutants revealed that the presence of at least two of these proteins depends on the activity of loci O, P, and Q of the Y chromosome. However, preliminary evidence indicates an autosomal location of the genes of these sperm proteins. This suggests a regulatory role of Y chromosomal genes in the production of some major sperm proteins.

Genetic and cytogenetic analysis of the "Th-Ps" region of the Y chromosome of Drosophila hydei: evidence for dual functions of the lampbrush loop-forming fertility genes?

Two competing hypotheses have been proposed for the function of the Y chromosomal fertility factors in Drosophila, which form giant lampbrush loops during the primary spermatocyte stage. The first hypothesis suggests a conventional coding function, the second proposes an unconventional gene function mediated through protein binding by nascent transcripts. Therefore, we studied the genetics and cytogenetics of the two Y chromosomal fertility genes A and C of Drosophila hydei (which form the lampbrush loops threads and pseudonucleolus) in order to test the validity of these different hypotheses. Both lampbrush loops bind specific proteins, which are recognized by different antisera. Absence of either of the lampbrush loops does not interfere with the synthesis of the antigens but completely prevents the binding of the particular antigen to other lampbrush loops. Absence of the loops also does not interfere with the postmeiotic presence and localization of the particular antigen. Deletion (or inactivation) of either of the lampbrush loops threads or pseudonucleolus causes sterility of the male flies as do other male-sterile alleles of both fertility genes, which do not affect the morphology of the lampbrush loops. The phenotypic effects of these mutations on sperm morphogenesis are identical for all various male-sterile alleles of each of the fertility genes A and C, regardless of whether a particular allele leaves the loop intact, modifies that loop, or deletes (or inactivates) the loop completely. Finally, the isolation of fertile Y chromosomal mutations which modify the morphology of the lampbrush loops demonstrates that it is possible to uncouple loop morphology and genetic function. These findings do not support the hypothesis that the binding of proteins to a lampbrush loop has a substantial impact on spermiogenesis.

Genetic versus histological grading in stereotactic biopsies.

With diagnostic stereotactic biopsies in astrocytoma sometimes false results will be encountered. A false positive result is defined as a histological diagnosis which is afterwards proved to be untrue. Although this happens very seldom (1-2%) in characterizing the tumor type (e.g. astrocytoma versus lymphoma), there is a probability of undergrading in malignant astrocytomas due to regional heterogeneity in malignancy grade. The issue of 'sampling error' has been addressed by many authors. It was shown that undergrading in astrocytomas is a likely event in fibrillar astrocytomas that often show geographically distinct areas of well and poorly differentiated elements. In a recent study on histological grading versus genetic characterization of heterogeneous astrocytomas we published that low-grade areas within high-grade malignant astrocytomas have already the genetic features of high-grade malignancy. Therefore, particularly in stereotactic biopsies which contain relatively small samples of tumor tissue, this fact is of paramount importance. Undergrading may lead to withholding radiotherapy from the patient and to falsification of long-term survival results in series of so-called low-grade astrocytomas. We like to stress the importance of taking biopsies from different parts of the tumor, especially from parts with different density enhancement to contrast with CT scanning, and will discuss the technical advancements made in the genetic grading of astrocytomas. Particularly loss of heterozygosity of chromosome 10 and eventually amplification of the EGFR indicate a high grade of malignancy.

Amplification of the anonymous marker D17S67 in malignant astrocytomas.

Loss of heterozygosity (LOH) for chromosome arms 9p, 10p, 10q, and 17p and amplification of the epidermal growth factor receptor (EGFR) gene have been identified as frequent genetic changes in malignant astrocytomas. We have found amplification of the anonymous marker D17S67 on chromosome arm 17p in 10% (3 of 30 cases) of astrocytomas of the highest malignancy grade. The tumors with D17S67 amplification displayed other genetic changes on chromosome 17, including additional amplifications and deletions. All three patients with D17S67 amplification developed severe brain edema and died within 1 month after operation.