Molecular genetic analysis of the NF2 gene in young patients with unilateral vestibular schwannomas

Neurofibromatosis type 2 (NF2) must be suspected in patients presenting with a unilateral vestibular schwannoma at a young age who are therefore at theoretical risk of developing bilateral disease. We identified 45 patients aged 30 years or less at the onset of symptoms of a unilateral vestibular schwannoma. Molecular genetic analysis of the NF2 gene was completed on peripheral blood samples in all 45 and on 28 tumour samples. No pathogenic NF2 mutations were identified in any of the blood samples. NF2 point mutations were identified in 21/28 (75%) tumour samples and loss of heterozygosity (LOH) in 21/28 (75%) tumour samples. Both mutational hits were identified in 18/28 (65%) tumour samples. In one multilobular tumour, one (presumably first hit) mutation was confirmed which was common to different foci of the tumour, while the second mutational event differed between foci. The molecular findings in this patient were consistent with somatic mosaicism for NF2 and the clinical diagnosis was confirmed with the presence of two meningiomas on a follow up MRI scan. A further patient developed a contralateral vestibular schwannoma on a follow up MRI scan in whom neither of the truncating mutations in the vestibular schwannoma were present in blood.

It is important when counselling patients with unilateral vestibular schwannomas to identify (1) those at risk of bilateral disease, (2) those at risk of developing other tumours, and (3) other family members at risk of developing NF2. Comparing tumour and blood DNA cannot exclude mosaicism in the index case and cannot, therefore, be used to predict those at risk of developing further tumours. However, identification of both mutations or one mutation plus LOH in the tumour and exclusion of those mutations in the blood samples of the sibs or offspring of the affected case may be sufficient to render further screening unnecessary in these relatives.

     

Mutations of the neurofibromatosis type 2 gene and lack of the gene product in vestibular schwannomas

Schwannomas are common tumors of the nervous system and arefrequently found in patients with neurofibromatosis (NF) 2.Although loss of heterozygosity in NF2 tumors suggests thatthe NF2 gene functions as a tumor suppressor gene, the NF2 geneshows amino acid sequence homology to structural proteins inone of which dominantly acting mutations have been described.We performed a mutational analysis in 30 vestibular schwannomasand examined the effect of mutations on the NF2 protein. Wedetected 18 mutations in 30 vestibular schwannomas of whichseven contained loss or mutation of both NF2 alleles. Most mutationswere predicted to result in a truncated protein. Mutationalhot spots were not identified. Immunocytochemical studies usingantibodies to the NF2 protein showed complete absence of stainingin tumor Schwann cells, whereas staining was observed in normalvestibular nerve. These data indicate that loss of NF2 proteinfunction is a necessary step in schwannoma pathogenesis andthat the NF2 gene functions as a recessive tumor suppressorgene.     

Differential diagnosis of type 2 neurofibromatosis: molecular discrimination of NF2 and sporadic vestibular schwannomas.

Patients who present with unilateral vestibular schwannomas either at a young age or with additional features of type 2 neurofibromatosis (NF2) are at risk of developing bilateral disease and transmitting a risk of neurogenic tumours to their offspring. We have identified 15 patients from a series of 537 with unilateral vestibular schwannomas who also had one or more of the following: other tumours (10/15), features of NF2 (3/15), or a family history of neurogenic tumours (5/15). No germline NF2 mutations were detected and in 7/9 cases where tumour material was available for analysis a germline mutation in the NF2 gene has been excluded. Although a possibility of gonosomal mosaicism still exists, exclusion tests for the offspring are now possible. We suggest a general strategy, based on analysis of tumour DNA, for distinguishing sporadic and familial cases of tumours caused by two hit mechanisms. Application of this strategy suggests that most instances of unilateral vestibular schwannoma which do not fulfil criteria for NF2 represent chance occurrences.     

Determination of the mutant allele frequency in patients with neurofibromatosis type 2 and somatic mosaicism by means of deep sequencing

Neurofibromatosis Type 2 (NF2) is an autosomal disorder caused by mutations of the NF2 gene. More than half of all NF2 patients have unaffected parents and carry de novo mutations, which may be of prezygotic or postzygotic origin. The latter can result in mosaicism, which is relatively common in NF2 patients. Previous studies indicated that, in 50% of patients with mosaic NF2 mutations, the mutant allele is only detectable by Sanger sequencing of PCR products amplified from tumor tissue but not from blood samples. In order to establish a highly sensitive method that has the power to detect low levels of NF2 mutant alleles from blood samples of mosaic NF2 patients, we performed ultra deep sequencing and calculated the percentage of mutant and wildtype NF2 alleles. The mutant allele frequencies detected ranged from 2.6% to 19.7%. In three patients, however, the NF2 mutation previously identified in tumor tissue was not identified in blood samples by means of deep sequencing, suggesting absence of mutant cells in the blood. Remarkably, we observed a correlation between the age at onset of the disease and the mutant allele frequency. Our study indicates that ultra deep sequencing is an effective and highly sensitive method to determine the mutant allele frequency in patients with mosaic NF2 gene mutations, which enables extended phenotype/correlations in these patients. © 2015 Wiley Periodicals, Inc.     

The neurofibromatosis type 2 gene is inactivated in schwannomas

Schwannomas are tumors arising from schwann cells surroundingperipheral nerves. Although most schwannomas are sporadic, theyare seen in approximately 90% of individuals with neuro-fibromatosistype 2 (NF2), an autosomai dominantiy inherited disease withan incidence of 1: 40000 live births. The NF2 gene has recentlybeen isolated on chromosome 22 and encodes a putative membraneorganizing protein named schwannomin. It is believed to actas a tumor suppressor gene based on the high frequency of lossof heterozygosity (LOH) on this autosome in both sporadic andNF2 associated schwannomas and meningiomas and the identificationof inactivating mutation in NF2 patients. In this study we examined61 schwannomas Including 48 sporadic schwannomas (46 of whichare vestlbular schwannomas) and 12 schwannomas obtained fromNF2 patients, for mutations in 10 of the 16 coding exons ofthe NF2 gene. Twelve inactivating mutations were Identified,8 In sporadic tumours and 4 in tumors from people with NF2.These results support the hypothesis that loss of function ofschwannomin is a frequent and fundamental event in the genesisof schwannomas.     

Effects of electrotactile vestibular substitution on rehabilitation of patients with bilateral vestibular loss

The present study evaluated the effectiveness of electrotactile tongue biofeedback (BrainPort^®) as a sensory substitute for the vestibular apparatus in patients with bilateral vestibular loss (BVL) who did not have a good response to conventional vestibular rehabilitation (VR). Seven patients with BVL were trained to use the device. Stimulation on the surface of the tongue was created by a dynamic pattern of electrical pulses and the patient was able to adjust the intensity of stimulation and spatially centralize the stimulus on the electrode array. Patients were directed to continuously adjust head orientation and to maintain the stimulus pattern at the center of the array. Postural tasks that present progressive difficulties were given during the use of the device. Pre- and post-treatment distribution of the sensory organization test (SOT) composite score showed an average value of 38.3 ± 8.7 and 59.9 ± 11.3, respectively, indicating a statistically significant improvement (p = 0.01). Electrotactile tongue biofeedback significantly improved the postural control of the study group, even if they had not improved with conventional VR. The electrotactile tongue biofeedback system was able to supply additional information about head position with respect to gravitational vertical orientation in the absence of vestibular input, improving postural control. Patients with BVL can integrate electrotactile information in their postural control in order to improve stability after conventional VR. These results were obtained and verified not only by the subjective questionnaire but also by the SOT composite score. The limitations of the study are the small sample size and short duration of the follow-up. The current findings show that the sensory substitution mediated by electrotactile tongue biofeedback may contribute to the improved balance experienced by these patients compared to VR.     

Mental transformation abilities in patients with unilateral and bilateral vestibular loss

Vestibular information helps to establish a reliable gravitational frame of reference and contributes to the adequate perception of the location of one’s own body in space. This information is likely to be required in spatial cognitive tasks. Indeed, previous studies suggest that the processing of vestibular information is involved in mental transformation tasks in healthy participants. In this study, we investigate whether patients with bilateral or unilateral vestibular loss show impaired ability to mentally transform images of bodies and body parts compared to a healthy, age-matched control group. An egocentric and an object-based mental transformation task were used. Moreover, spatial perception was assessed using a computerized version of the subjective visual vertical and the rod and frame test. Participants with bilateral vestibular loss showed impaired performance in mental transformation, especially in egocentric mental transformation, compared to participants with unilateral vestibular lesions and the control group. Performance of participants with unilateral vestibular lesions and the control group are comparable, and no differences were found between right- and left-sided labyrinthectomized patients. A control task showed no differences between the three groups. The findings from this study substantiate that central vestibular processes are involved in imagined spatial body transformations; but interestingly, only participants with bilateral vestibular loss are affected, whereas unilateral vestibular loss does not lead to a decline in spatial imagery.     

Expression pattern of apoptotic markers in vestibular schwannomas

The Fas-Fas-L system plays a major role in the regulation of apoptosis and hence in growth in benign and malignant human tumors. As the factors regulating cell death in benign schwannomas are not well understood, we investigated the immunoexpression of the Fas-Fas-L system, as well as that of the anti-apoptotic factor Bcl-2 and the pro-apoptotic factor Bax in 14 sporadic vestibular schwannomas, and related the findings to the MIB-1 labeling index as a marker for cell proliferation. Whereas cytoplasmic Fas expression was seen in only one tumor (7%), Fas-L was found in the nuclei of 12 schwannomas (86%). Bcl-2 expression was found in the cytoplasm of 9 tumors (64%), and Bax was found in 10 out of 14 schwannomas (71%). No significant correlations between different labeling indices were observed. However, schwannomas expressing Bax tended to show a higher proliferation rate as revealed by the MIB-1 LI, suggesting a balance between cell proliferation and cell death. Our study further showed that Fas-L is present in most vestibular schwannomas; however, due to the lack of Fas expression, apoptosis in vestibular schwannomas does not seem to be mediated via the Fas-Fas-L system.     

High frequency of tissue-specific mosaicism in Turner syndrome patients

Interphase fluorescent studies of X chromosome aneuploidy in cultured and uncultured blood lymphocytes and oral mucosa epithelial cells using X centromere-specific DNA probe in addition to standard karyotype analysis were performed in 50 females with a clinical suspicion of Turner syndrome. All the patients were previously screened for the presence of 'hidden' Y chromosome mosaicism, using the primers DYZ3 and DYZ. The use of fluorescence in situ hybridization (FISH) analysis of interphase nuclei of tissues from different germ layers (lymphocytes from mesoderm and buccal epithelial cells from ectoderm) improves the accuracy of detection of low-level mosaicism. FISH studies on interphase nuclei revealed that 29% of patients with a pure form of monosomy X detected by metaphase analysis are, in fact, mosaics. The level of cells with the normal chromosomal constitution in lymphocytes of these cases as a rule was low, ranging from 3 to 18%, with an average of 7%. Two false-positive cases and one false-negative case of X monosomy mosaicism determined by standard cytogenetic approach were detected using FISH analysis. The majority of patients (92%) with mosaic form of Turner syndrome have considerable tissue-specific differences in levels of X aneuploidy. Our data indicate that in cases when mosaic aneuploidy with low-level frequency is questionable (approximately 10% and lower), the results of standard metaphase analysis should be supplemented with additional FISH studies of interphase nuclei. Tissue-specific differences in contents of different cell lines in the same patients point to the necessity of studying more than one tissue from each patient.     

Molecular genetic analyses in neurofibromatosis type 1 patients with tumors

Neurofibromatosis type 1 (NF1) is one of the most common autosomal dominant disorders. NF1 is clinically characterized by neurofibromas, pigmentation anomalies, and an increased risk of malignant tumors. The NF1 gene product, neurofibromin, has a GTPase-activating protein domain (GRD) that interacts with the Ras protein, which is crucial in regulating signal transduction and cell proliferation/differentiation. We performed mutation analyses in the NF1-GRD region (exons 21-27a) and in exons 4b, 16, 29, and 37, and intron 28 in 17 NF1 patients with tumors. We identified a large deletion in the NF1 gene in a patient with a rhabdomyosarcoma as well as a variation in intron 22 in a patient with an optic glioma. We also found a 4-base pair deletion in another patient with optic glioma. In addition, allelic loss of the NF1 locus was shown in a pilocytic astrocytoma. Functional analyses of mutations in the NF1 gene may provide further insights into the pathogenesis of NF1 tumors.     

Methodological issues in longitudinal studies: vestibular schwannoma growth rates in neurofibromatosis 2

Four longitudinal studies of vestibular schwannoma (VS) growth rates in neurofibromatosis 2 (NF2) have yielded very different results on the relationship of VS growth rates to age. The studies had different patient eligibility criteria, indices of VS growth rates, VS volumetric methods, and sample sizes. We reanalysed data from two of the longitudinal studies and used data from the population based United Kingdom NF2 Registry to determine the most likely reason for the different results and the actual relationship of VS growth rates to age. We found that the eligibility criterion in one study caused selection bias for slower growing VS. The proper interpretation of the results from the four studies is that VS growth rates in NF2 are highly variable but tend to decrease with increasing age. Clinical trials for VS in NF2 should focus on younger patients because VS growth rates tend to decrease with increasing age, and because faster growing VS are more likely to be excised with increasing age than slower growing VS.     

Interphase-FISH study in three patients with tetraploid/diploid mosaicism

We report tetraploid/diploid mosaicism in a boy and two girls detected by cultured skin fibroblasts. In all these children, interphase-FISH using DXZ1 probe confirmed the original karyotype. Tetraploid mosaicism was also confirmed by alpha-satellite probes from chromosomes 2, 6, 7, 9, 10, 17, 18 and Y in fibroblasts from the boy. The common features in these children are failure to thrive, slight mental deficiency and some degree of body asymmetry. The advantage of using interphase-FISH is the possibility of analysing a great number of cells in short time, thus giving a more precise percentage with regard to abnormal cells.     

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.      

Optically induced plasticity of the cervico-ocular reflex in patients with bilateral absence of vestibular function

The horizontal cervico-ocular reflex (COR) was examined in five labyrinthine-defictive subjects (LDS), during both passive oscillations of the head on the trunk (HTexam) and of the trunk under the earthfixed head (THexam) at 0.1–0.5 Hz, peak angular displacement ±30°. Subjects were tested in the dark, before and immediately after adaptation to binocular magnifying (x1.9) and reducing (x0.6) lenses. During long-term adaptation, the LDS were exposed to the normal environment for 5 h while wearing lenses. Short-term adaptation experiments (15–20 min) consisted of sustained ocular following of a small LED in an otherwise dark room and in full-room illumination. This LED was either stationary in space whilst the subjects moved their head actively, or fixed on the chair and rotating with the trunk during head-fixed stimulation. In all five patients, magnifying lenses increased COR gain (peak slow-phase eye velocity/peak stimulus velocity), whereas reducing lenses reduced the gain. Under HTexam the gain changes were greater, more consistent and the phases approximately compensatory to head displacement, whereas during THexam the gain decreased and phase increased at higher frequencies. COR adaptation was observed during foveal stimulation alone, but the effects were stronger with added background illumination. Results during an imaginary target task showed that the gain can be influenced strongly by mental set. Our findings indicate a highly modifiable COR in subjects with loss of vestibular function. Both peripheral and foveal retinal information contribute to the plastic changes in COR gain. Somatosensory cues from the trunk as well as cognitive/perceptual factors may be involved in the modification of the COR, by providing information about the relevance of eye movements, and contribute to the stabilisation of gaze in space.     

NF2 gene in neurofibromatosis type 2 patients

Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder that predisposes to nervous system tumors. The schwannomin (also termed merlin) protein encoded by the NF2 gene shows a close relationship to the family of cytoskeleton-to-membrane proteins linkers ERM (ezrin- radixin-moesin proteins). Even though penetrance of the disease is >95% and no genetic heterogeneity has been described, point mutations in the NF2 gene have been observed in only 34-66% of the screened NF2 patients, depending on the series. In order to generate tools that would enable an exhaustive alteration screening for the NF2 gene, we have deduced its entire genomic sequence. This knowledge has provided the delineation of a mutation screening strategy which, when applied to a series of 19 NF2 patients, has revealed a high recurrence of large deletions in the gene and has raised the efficiency of mutation detection in NF2 patients to 84% of the cases in this series. The remaining three patients who express two functional NF2 alleles are all sporadic cases, an observation compatible with the presence of mosaicism for NF2 mutation.