Neurofibromatosis 2 Patients Research Articles

Pediatric vestibular schwannoma with or without neurofibromatosis: Characteristic features and operative nuances

Vestibular schwannomas (VSs) are rarer in children than in adults and are often associated with neurofibromatosis 2 (NF2). We focus on characteristic presentations of VSs in children (< 16 years old) both with and without NF2 and illustrate essential surgical steps using the retromastoid suboccipital approach. <b>Materials and Methods:</b> Three patients had unilateral tumors and five had bilateral tumors with associated NF2. On computed tomography (CT)/ magnetic resonance (MR) imaging, tumor diameters were measured parallel and perpendicular to the petrous ridge and vertically in coronal slices. The size of the VS was taken as the largest diameter in any one axis. Tumors were categorized as: Small: < 1 cm in size; Medium: 1-2.5 cm; Large: 2.5-4 cm and Giant: > 4 cm. Surgery was done in the park-bench position using the suboccipital retromastoid approach. <b>Results:</b> Raised intracranial pressure (ICP), often with visual deterioration, was the most commonly found presentation. Preoperative VIIth nerve involvement was grade IV in three, III in two and II in two patients. One of the patients with NF2 with proptosis did not have any VIIth nerve deficit. None of the patients had any functionally useful hearing on the side of larger tumor. Tumors were giant in six and large in two patients. In NF2 patients, two patients had large, two had medium-sized and one patient had a small contralateral tumor. Seven patients had significant hydrocephalus out of which three underwent ventriculoperitoneal shunt. Total excision of the larger lesion was performed in six patients. A small tumor remnant was left adherent to the preserved VIIth nerve in one patient. A patient with NF2 underwent excision of orbital neurofibroma and is awaiting surgery for VS. Two patients underwent bilateral VS excision. Anatomical preservation of the VIIth nerve was possible in six out of seven patients who underwent tumor excision. Postoperatively, symptoms of raised intracranial pressure and ataxia improved; visual deficits continued to deteriorate in two and VIIth nerve function deteriorated from grade III to V. <b>Conclusions:</b> In children, only a high degree of awareness facilitates early diagnosis of VS; otherwise, initial complaints may be neglected and the underlying tumor detected only after incapacitating raised ICP features appear. The retromastoid suboccipital approach is an effective approach for safe removal of large to giant-sized tumors.

Uptake of 4-borono-2-[18F]fluoro-L-phenylalanine in sporadic and neurofibromatosis 2-related schwannoma and meningioma studied with PET

Meningiomas and schwannomas associated with neurofibromatosis 2 (NF2) are difficult to control by microsurgery and stereotactic radiotherapy alone. Boron neutron capture therapy (BNCT) is a chemically targeted form of radiotherapy requiring increased concentration of boron-10 in tumour tissue. PET with the boron carrier 4-borono-2-[(18)F]fluoro-L-phenylalanine ([(18)F]FBPA) allows investigation of whether 4-borono-L-phenylalanine (BPA) concentrates in NF2 tumours, which would make BNCT feasible. We studied dynamic uptake of [(18)F]FBPA in intracranial meningiomas (n=4) and schwannomas (n=6) of five sporadic and five NF2 patients. Tracer input function and cerebral blood volume were measured. [(18)F]FBPA uptake in tumour and brain was assessed with a three-compartmental model and graphical analysis. These, together with standardised uptake values (SUVs), were used to define tumour-to-brain [(18)F]FBPA tissue activity gradients. Model fits with three parameters K (1) (transport), k (2) (reverse transport) and k (3) (intracellular metabolism) were found to best illustrate [(18)F]FBPA uptake kinetics. Maximum SUV was two- to fourfold higher in tumour as compared with normal brain and independent of NF2 status. The increased uptake was due to higher transport of [(18)F]FBPA in tumour. In multiple-time graphical analysis (MTGA, Gjedde-Patlak plot) the tumour-to-brain [(18)F]FBPA influx constant (K (i) -MTGA) ratios varied between 1.8 and 5.4 in NF2-associated tumours while in sporadic tumours the ratio was 1-1.4. [(18)F]FBPA PET offers a viable means to evaluate BPA uptake in meningiomas and schwannomas in NF2. Based on our results on tumour uptake of [(18)F]FBPA, some of these benign neoplasms may be amenable to BNCT.

Neurofibromatosis 2 leads to choroidal hyperfluorescence in fluorescein angiography

For the diagnosis of NF 2, ocular findings like juvenile cataract, retinal and combined hamartomas of the retinal pigment epithelium and the retina as well as tumours of the optic nerve play an important role. An early diagnosis is essential in order to inhibit deafness from bilateral vestibular schwannoma. But sometimes the Manchester diagnostic criteria for NF2 are not completely fulfilled. Frequently, suspicious macular anatomy is found in neurofibromatosis 2 (NF2) patients. We hypothesise that the underlying retinal pigment epithelium or the retina of the macular region alters in NF2 patients. Therefore, we have tested by fluorescence angiography whether NF2 is associated with chorioretinal changes. In a prospective study, 48 patients matching the criteria for NF2 with known genotype underwent a complete ophthalmic examination including funduscopy and fluorescence angiography. The influence of the genotype was statistically analysed by odds ratios and their 95% confidence intervals. Eleven eyes of nine patients showed choroidal hyperfluorescence in the macular region on fluorescence angiography. There was staining spreading from grainy hyperfluorescence to minor variants of a combined hamartoma of the retina and the retinal pigment epithelium. All of these manifestations presented without leakage in the late angiographic phases. These choroidal findings were present in one patient with frameshift mutation, in two patients with nonsense mutations and in six patients with splice site mutations of the NF2 gene. The statistical analysis showed a significant lower risk of choroidal alterations in patients with somatic mosaicism, deletions and unfound mutations. Using fluorescence angiography pathological changes of the macular region can be detected in NF2 patients. The ophthalmic examination, which often is limited to the anterior eye segment, may play a role in finding the diagnosis in incomplete NIH criteria. The presented study shows chorioretinal hyperfluorecences without leakage of the macular region, which might be considered as a forme fruste of a hamartoma. Choroidal hyperfluorescences add to the spectrum of genotype-phenotype correlations in NF2.

Schwannomin inhibits tumorigenesis through direct interaction with the eukaryotic initiation factor subunit c (eIF3c)

The neurofibromatosis 2 (NF2) tumor suppressor protein, schwannomin or merlin, is commonly lost upon NF2 gene mutation in benign human brain tumors. We identified the p110 subunit of the eukaryotic initiation factor 3 (eIF3c) as a schwannomin interacting protein. The eIF3 complex consists of approximately 10 subunits whose functions are only recently becoming known. Interaction between schwannomin and eIF3c suggests a role for schwannomin in eIF3c-mediated regulation of proliferation related to changes in protein translation. We found that schwannomin was most effective for inhibiting cellular proliferation when eIF3c was highly expressed. When we examined these proteins in 14 meningiomas, we observed high eIF3c abundance in those that had lost schwannomin expression but low eIF3c abundance in those retaining schwannomin. Consequently, eIF3c appears to be involved in NF2 pathogenesis and deserves to be investigated as a prognostic marker for NF2 and target for treatment of NF2 patient tumors.

Screening for large mutations of the NF2 gene

Neurofibromatosis 2 (NF2) is a genetic disorder caused by mutational inactivation of the NF2 gene and is characterized by bilateral vestibular schwannomas, spinal tumors, and other benign tumors of the nervous system. Previously, we found intragenic NF2 mutations in 99 of 188 unrelated NF2 patients by exon-scanning-based methods. Tumor analysis of 22 de novo NF2 patients led to the identification of 12 additional constitutive NF2 mutations. The remaining 77 patients were further examined for large alterations using the newly developed gene dosage assay multiplex ligation-dependent probe amplification (MLPA). One deletion of a single exon, seven deletions of multiple exons, seven deletions involving the 3' or 5' end of the NF2 gene, four deletions involving the whole NF2 gene, and one duplication of three exons were detected. For 47 of the 77 patients, mRNA of adequate quality could be obtained, enabling transcript analysis, which confirmed eight alterations detected by MLPA. In addition, in one family, the mRNA analysis detected an insertion of two exons of another gene. Thus, deletions, duplications, and insertions affecting the NF2 gene were found in 21 cases, which is 11% of the 188 unrelated NF2 patients studied, 16% of the 132 mutations identified, and 27% of the 77 cases in which no intragenic small mutations were detected by exon scanning. The combination of multiple screening techniques facilitated a mutation-detection rate of 100% for the 21 inherited cases in this study.

ABR change over time in neurofibromatosis 2

Objectives: To determine whether clinically obtained auditory brainstem response (ABR) shows significant changes over time associated with the growth of vestibular schwannomas related to neurofibromatosis 2 (NF2) and to evaluate the clinical use of derived band ABR in NF2 patients. Methods: Subjects meeting the NIH NF2 diagnostic criteria were enrolled (n = 103) across 9 domestic and international investigational sites. ABR and MRI exams were conducted annually. Each site was instructed to follow a comprehensive protocol for acquiring the MRI. The sites were instructed to assess the ABR either with the usual clinical parameters or using derived band methodology. Results: Wave 5 latency data were available for 35 subjects at both baseline and year 1. Average latency values were found to be clinically normal at both evaluations (average = 6.4 ms). Subjects experienced an average change in Wave 5 from baseline to year 1 of 0.06 ms. A strong association was found between Wave 5 latency at baseline and the greatest diameter measurement of the tumor (Pearson’s correlation = 0.62, P < 0.05), where larger tumors have longer latencies. The subjects with derived band ABR were further evaluated for change over time. Conclusions: ABR wave 5 latency shows a strong association with size, the longer the latency, the larger the vestibular schwannoma, regardless of how the ABR was obtained. Across evaluations, there was a nonsignificant change in wave latencies. Clinically obtained ABR provides information regarding size but provides less information about the change in vestibular schwannomas over time. Derived band ABR offers greater measurement specificity for observing NF2 patients over time.

Differentially expressed genes in neurofibromatosis 1-associated neurofibromas and malignant peripheral nerve sheath tumors

Neurofibromas represent one of the hallmarks of neurofibromatosis 1 (NF1) patients. Tumor progression of neurofibromas to malignant peripheral nerve sheath tumors (MPNST) is a frequent and life threatening complication. To learn more about processes involved in malignant transformation, we evaluated differential gene expression in plexiform neurofibroma and MPNST from the same NF1 patient. Suppression subtractive hybridization (SSH) yielded 133 differentially expressed genes confirmed by reverse Northern blotting. Virtual Northern blots were employed to validate 23 genes. To independently verify differential expression, immunohistochemical analyses with antibodies to matrix metalloproteinase 13 (MMP13), platelet-derived growth factor receptor alpha (PDGFRA) and fibronectin (FN1) were performed on 9 dermal and 9 plexiform neurofibromas and 16 MPNST from 19 NF1 patients. All three proteins proved to be up-regulated in MPNST. MMP13 expression was observed in 44% of MPNST but was absent in neurofibromas. PDGFRA was expressed in all tumors, but the number of cells expressing it was below 30% in neurofibromas and over 50% in MPNST. Likewise, FN1 was expressed in all tumors, but less than 30% of the cells in neurofibromas and more than 70% of the cells in MPNST exhibited antibody binding. Our data point to several genes not previously recognized to be differentially expressed, and provide a framework for future studies on progression-associated gene expression in low- and high-grade nerve sheath tumors.

Exploring the "two-hit hypothesis" in NF2: tests of two-hit and three-hit models of vestibular schwannoma development.

Neurofibromatosis 2 (NF2) is a genetic disease that occurs in approximately 1 in 40,000 live births. Almost all affected individuals develop bilateral tumors of Schwann cells that surround the vestibular nerves; these tumors are known as vestibular schwannomas (VS). Evidence from molecular genetic studies suggests that at least two mutations are involved in formation of VS in patients with NF2. Several authors proposed probabilistic models for this process in other tumors, and showed that such models are consistent with incidence data. We evaluated two different probabilistic models for a "2-hit" hypothesis for VS development in NF2 patients, and we present results from fitting these models to incidence data. Molecular evidence does not exclude the possibility that additional hits are necessary for the development of VS, and we also assessed a "3-hit" model for tumor formation. The "3-hit" model fits the data marginally better than one of the "2-hit" models and much better than the other "2-hit" model. Our findings suggest that more than two mutations may be necessary for VS development in NF2 patients.

Neurofibromatosis 2.

Recent clinical and molecular research on neurofibromatosis 2 (NF2) is reviewed, and the implications for clinical practice and research are discussed. NF2 patients who are treated in specialty centers have a significantly lower risk of mortality than those who are treated in non-specialty centers. Vestibular schwannoma growth rates in NF2 are generally higher in younger people but are highly variable, even among multiple NF2 patients of similar ages in the same family. Radiation therapy is best reserved for NF2 patients who have particularly aggressive tumors, those who are poor surgical risks, those who refuse surgery, or those who are elderly. In-vivo studies have demonstrated that leptomeningeal cell activation of in mice results in leptomeningeal hyperplasia and meningioma formation. In-vitro studies have identified molecules that interact with the product (merlin or schwannomin), some of which (e.g., CD44 and paxillin) may play critical roles in merlin growth regulation. NF2 patients should be referred to specialty treatment centers for optimal care. Clinical management of multiple patients in NF2 families cannot be based on the expectation of similar vestibular schwannoma growth rates, even when other clinical aspects of disease severity are similar. The availability of accurate mouse models of human NF2-associated tumors and the identification of molecules involved in merlin growth regulation now provide an opportunity to design targeted treatments for schwannomas and meningiomas.

Loss of heterozygosity for the NF2 gene in retinal and optic nerve lesions of patients with neurofibromatosis 2.

Individuals affected with the neurofibromatosis 2 (NF2) cancer predisposition syndrome develop specific ocular lesions. To determine whether these lesions result from altered NF2 gene expression, microdissection and PCR were used to investigate 40 ocular lesions from seven eyes of four NF2 patients for LOH, with markers that flank the NF2 gene on chromosome 22q. NF2 protein (merlin) expression was also evaluated in these lesions, using immunohistochemistry. Retinal hamartoma was observed in all seven eyes, including one with combined pigment epithelial and retinal hamartoma (CPERH). Retinal tufts were present in four eyes (three patients), retinal dysplasia in two eyes (two patients), optic nerve neurofibroma in one eye, iris naevoid hyperplasia in two eyes (two patients) and pseudophakia in all eyes. Markers were informative in three patients (six eyes from three unrelated families). One patient was non-informative due to prolonged decalcification. All retinal and optic nerve, but not iris lesions, demonstrated consistent LOH for the NF2 gene. Merlin was not expressed in the retina, optic nerve, or iris lesions. These results suggest that inactivation of the NF2 gene is associated with the formation of a variety of retinal and optic nerve lesions in NF2 patients.

Malignant peripheral nerve sheath tumours in neurofibromatosis 1

Background: Cross sectional studies have shown that 1-2% of patients with neurofibromatosis 1 (NF1) develop malignant peripheral nerve sheath tumours (MPNST). However, no population based longitudinal studies have assessed lifetime...

Polyneuropathy in neurofibromatosis 2: clinical findings, molecular genetics and neuropathological alterations in sural nerve biopsy specimens.

Neurofibromatosis 2 (NF2) is an autosomal dominant disease characterised by development of tumours in the central and peripheral nervous system. Some NF2 patients develop acro-distal sensory motor polyneuropathy that can hardly be explained by the tumour burden alone. In the present study eight sural nerve biopsy specimens from seven NF2 patients suffering from polyneuropathy were investigated, data including clinical course of the disease, electrophysiological findings, teased fibre preparations, histopathological, morphometric, immunohistochemical, electron microscopic and molecular genetic findings. All patients suffered from distal symmetric reflex loss, symmetrical stocking-like hypalgesia and hypesthesia and loss of vibration sense later followed by a slowly progressive distal muscle atrophy and paresis. Sural nerve biopsy specimens revealed a pathological reduction of nerve fibre density correlating with age. In addition, diffuse proliferation of Schwann cells was observed in five of eight biopsies, and small endoneurial tumourlets of schwannomas and perineuriomas were found in two of eight and one of eight samples, respectively. Ki-67 labelling revealed a slight endoneurial proliferative activity in three cases. Schwann cell onion bulbs with or without central myelinated axon were seen in two cases. The findings suggest an axonopathy of multifactorial origin resulting not only from gross tumour growth but, in addition, from small endoneurial tumourlets, diffuse proliferation of Schwann cells and proliferation of perineurial cells.

The parental origin of new mutations in neurofibromatosis 2.

Neurofibromatosis 2 (NF2) is an autosomal dominant disorder characterized by schwannomas and meningiomas that develop after inactivation of both copies of the NF2 gene. Approximately half of all patients with NF2 have unaffected parents and the disease results from new mutations at the NF2 locus. Loss of heterozygosity (LOH) in tumor specimens due to deletions covering the normal NF2 allele can be used to infer the haplotypes surrounding underlying mutations and determine the allelic origin of new mutations. We studied 71 sporadic NF2 patients using both LOH and pedigree analysis and compared the parental origin of the new mutation with the underlying molecular change. In the 45 informative individuals, 31 mutations (69%) were of paternal and 14 (31%) were of maternal origin (P=0.016). Comparison with corresponding constitutional mutations revealed no correlation between parental origin and the type or location of the mutations. However, in 4 of 6 patients with somatic mosaicism the NF2 mutation was of maternal origin. A slight parent of origin effect on severity of disease was found. Further clinical and molecular studies are needed to determine the basis of these unexpected observations.

A clinical study of vestibular schwannomas in type 2 neurofibromatosis.

All 13 patients with neurofibromatosis 2 (NF2) who presented over a period of 17 years at the Institute of Neurological Sciences, Glasgow were reviewed and compared to patients with sporadic vestibular schwannomas. The NF2 patients presented at a younger age than those with sporadic vestibular schwannomas. A significant number had normal pure tone audiograms and a small number also had normal auditory brainstem responses at presentation. Vestibular schwannomas in NF2 patients grow more often and more rapidly than sporadic unilateral ones. They are more liable to infiltrate the cochlear and facial nerves making hearing and facial nerve preservation more difficult to achieve. Because the relatives of these patients often have normal audiograms and normal auditory brain stem responses in the presence of a schwannoma, our recommended method of screening of relatives of NF2 patients is magnetic resonance image scanning with Gd-DTPA enhancement.

Allelic losses in neurofibromatosis 2-associated meningiomas.

More than 50% of patients with neurofibromatosis 2 (NF2) develop meningiomas. Recently, a higher proliferative activity, more mitotic figures, and greater nuclear pleomorphism have been described for NF2-associated meningiomas compared with sporadic ones. To analyze whether such histological differences could reflect underlying genetic differences, we examined 30 meningiomas from 22 NF2 patients for allelic losses on those chromosome arms that are frequently affected by deletions in sporadic meningiomas. In addition, we assessed the proliferative activity of the tumors and studied NF2 germline mutations. Twenty-three meningiomas corresponded to WHO grade I (10 fibrous, 6 psammomatous, 4 transitional, 3 meningothelial) and 7 to WHO grade II. The average MIB-1 index was 1.60 +/- 0.85 (WHO grade I: 1.41 +/- 0.80, WHO grade II: 2.13 +/- 0.82). When compared with several published studies of sporadic meningiomas, the MIB-1 index in NF2-associated meningiomas was not higher. Loss of heterozygosity (LOH) flanking or within the NF2 locus at 22q12 was detected in 100% of the tumors. LOH on 1p was the second most frequent abnormality (40%), followed by losses on 10q (27%), 6q and 14q (24%), 18q (23%), and 9p (17%). LOH on 19q and 17p, which is not commonly seen in sporadic meningiomas, was also only rarely detected in NF2-associated meningiomas. NF2 gene mutations were detected in 8 of 15 patients analyzed and were located in exons 2, 5, 6, 7, and 8. We conclude that sporadic and NF2-associated meningiomas share a common spectrum and frequency of allelic deletions as well as, in contrast to previous observations, a similar proliferative activity.

Neurofibromatosis 2 tumor suppressor protein, merlin, forms two functionally important intramolecular associations

The neurofibromatosis 2 (NF2) tumor suppressor gene product, merlin (schwannomin) forms an intramolecular association that is required for negative growth regulation in vitro and in vivo. In an effort to develop a molecular model for merlin relevant to its tumor suppressor function, we further characterized merlin intramolecular folding. We now demonstrate that merlin forms two intramolecular associations, one between the amino terminal (N-term) domain and the carboxyl terminal (C-term) domain and another within the amino terminal domain (N-term/N-term) itself. The N-term/C-term domain interaction requires contact between residues 302-308 in the N-term and an intact exon 17 (residues 580-595) in the C-term domain. In addition, we demonstrate that the N-term/N-term domain self-interaction is required for N-term/C-term domain association. Lastly, we identify NF2 patient mutations that dramatically reduce each of these interactions in vitro. Based on these findings, we propose a model for merlin folding critical to its ability to function as a tumor suppressor protein.

Increased expression of the NF2 tumor suppressor gene product, merlin, impairs cell motility, adhesionand spreading.

The neurofibromatosis 2 ( NF2 ) gene product, merlin, is a tumor suppressor protein mutated in schwanno-mas and several other tumors. Merlin, which shares significant homology with the actin-associated proteins ezrin, radixin and moesin (ERM proteins), inhibits cell growth when overexpressed in cell lines. The similarities between merlin and ERM proteins suggest that merlin's growth-regulatory capabilities may be due to alterations in cytoskeletal function. We examined this possibility in rat schwannoma cell lines overexpressing wild-type merlin isoforms and mutant merlin proteins. We found that overexpression of wild-type merlin resulted in transient alterations in F-actin organization, cell spreading and cell attachment. Merlin overexpression also impaired cell motility as measured in an in vitro motility assay. These effects were only observed in cells overexpressing a merlin isoform capable of inhibiting cell growth and not with mutant merlin molecules (NF2 patient mutations) or a merlin splice variant (isoform II) lacking growth-inhibitory activity. These data indicate that merlin may function to maintain normal cytoskeletal organization, and suggest that merlin's influence on cell growth depends on specific cytoskeletal rearrangements.

Mosaicism in sporadic neurofibromatosis 2 patients.

More than half of neurofibromatosis 2 (NF2) patients represent de novo mutations which could have occurred at either pre-zygotic or post-zygotic stages. A post-zygotic mutation can result in mosaicism. In four sporadic NF2 patients, we found NF2 mutations in only a portion of corresponding leukocytes. In two other sporadic patients, no mutations were found in leukocytes but constitutional NF2 mutations were suggested by identical mutations in different tumors from each patient. We screened leukocyte DNA from a total of 16 inherited and 91 sporadic NF2 patients, and found NF2 mutations in 13 (81%) of the former and in 46 (51%) of the latter cases. The 30% difference in the rate of detection of mutations ( P = 0.051) might be partially explained by mosaicism in a portion of sporadic NF2 patients who carry the mutations in such a fashion that their leukocytes are unaffected. Among sporadic cases, we found mutations more frequently in patients with severe phenotypes (59%) than in patients with mild phenotypes (23%) (difference of 36%, P = 0.007). Mosaicism might be more common in the latter patient group since small populations of mutation-bearing cells can in some cases result in mild phenotypes and can also lead to difficulties in identifying mutations. No mutations were found in eight patients suspected of having NF2. Mosaicism with an extremely small population of affected cells may explain the incomplete phenotypes in some of these patients and the lack of mutations in their leukocytes. These findings suggest that mosaicism is relatively common in NF2 and may have important implications for diagnosis, prognosis and genetic counseling.

Defects in neurofibromatosis 2 protein function can arise at multiple levels.

Neurofibromatosis 2 (NF2) is an inherited cancer syndrome resulting from mutations in the NF2 tumor suppressor gene. Analysis of NF2 mutations has revealed some general genotype-phenotype correlations. Severe disease has been associated with mutations that produce a premature termination while more mild disease has been associated with missense mutations. Here, we provide experimental proof for these genotype-phenotype correlations by demonstrating that nonsense mutations fail to produce stable merlin protein while missense mutations result in the generation of merlin proteins defective in negative growth regulation. This inability to suppress cell growth may result from defects in the function of merlin at several levels, including failure to form an intramolecular complex. Based on these findings, we propose a model for merlin growth suppression that provides a framework for analyzing NF2 patient mutations and merlin function.

Merlin differentially associates with the microtubule and actin cytoskeleton.

The neurofibromatosis 2 (NF2) suppressor gene encodes a protein termed merlin (or schwannomin) with sequence similarity to a family of proteins that link the actin cytoskeleton to cell surface glycoproteins. Members of this ERM family of proteins include ezrin, radixin, and moesin. These proteins contain a carboxyl (C-) terminus actin binding site. In contrast to the ERM proteins, merlin lacks the conventional C-terminal actin binding site, but still localizes to the ruffling edge of plasma membranes. In this study, we investigate the ability of merlin to interact with actin through a nonconventional actin binding domain. We demonstrate for the first time that merlin can associate with polymerized actin in vitro by virtue of an amino (N-) terminal actin binding domain including residues 178-367. Merlin actin binding is not affected by several naturally-occurring NF2 patient mutations or alternatively spliced isoforms. These results suggest that merlin, like other ERM proteins, can directly interact with the actin cytoskeleton. In addition, merlin associates with polymerized microtubules in vitro using a novel microtubule binding region in the N-terminal region of merlin that is masked in the full-length merlin molecule, such that wild-type functional merlin in the "closed" conformation fails to bind polymerized microtubules. These microtubule association results confirm the notion that merlin exists in "open" and "closed" conformations relevant to its function as a negative growth regulator.