Search

Early onset of vestibular schwannoma (VS) is associated with the inherited condition neurofibromatosis type 2 (NF2). However, the majority of NF2 presents bilaterally and the proportion of early-onset apparent sporadic unilateral VS because of NF2 remains to be determined. We have determined the risk by studying NF2 risk in a population-based set of VS, looking at the mode of presentation in a large NF2 data set and the outcome of NF2 mutation analysis in 148 sporadic unilateral VS. The risk of NF2 in an apparently sporadic case of unilateral VS is small apart from in the very youngest age group (

Neurofibromatosis type 2 (NF2) is almost unique among inherited disorders in the frequency of mosaicism in the first affected generation. However, the implications of this on transmission risks have not been fully elucidated.The expanded database of 460 families with NF2 and 704 affected individuals was analysed for mosaicism and transmission risks to offspring.64 mosaic patients, with a projected mosaicism rate of 33% for sporadic classical NF2 with bilateral vestibular schwannoma at presentation and 60% for those presenting unilaterally, were identified. Offspring risks can be radically reduced on the basis of a sensitive mutation analysis of blood DNA including multiple ligation-dependent probe amplification (MLPA, which detects 15% of all mutations), but even MLPA cannot detect high levels of mosaicism.The chances of mosaicism in NF2 and the resultant risks of transmission of the mutation to offspring in a number of different clinical situations have been further delineated. The use of MLPA in this large NF2 series is also reported for the first time.

Heterozygous whole gene deletions (WGDs), and intragenic microdeletions, account for a significant proportion of mutations underlying cancer predisposition syndromes. We analyzed the frequency and genotype-phenotype correlations of microdeletions in 12 genes (BRCA1, BRCA2, TP53, MSH2, MLH1, MSH6, PMS2, NF1, NF2, APC, PTCH1, and VHL) representing seven tumor predisposition syndromes in 5,897 individuals (2,611 families) from our center. Overall, microdeletions accounted for 14% of identified mutations. As expected, smaller deletions or duplications were more common (12%) than WGDs (2.2%). Where a WGD was identified in the germline in NF2, the mechanism of somatic second hit was not deletion, as previously described for NF1. For neurofibromatosis type 1 and 2, we compared the mechanism of germline deletion. Unlike NF1, where three specific deletion sizes account for most germline WGDs, NF2 deletion breakpoints were different across seven samples tested. One of these deletions was 3.93 Mb and conferred a severe phenotype, thus refining the region for a potential NF2 modifier gene to a 2.04-Mb region on chromosome 22. The milder phenotype of NF2 WGDs may be due to the apparent absence of chromosome 22 loss as the second hit. These observations of WGD phenotypes will be helpful for interpreting incidental findings from microarray analysis and next-generation sequencing.

Background Germline mutations in BRCA1 or BRCA2 lead to a high lifetime probability of developing ovarian or breast cancer. These genes can also be involved in the development of non-hereditary tumours as somatic BRCA1/2 pathogenic variants are found in some of these cancers. Since patients with somatic BRCA pathogenic variants may benefit from treatment with poly ADP ribose polymerase inhibitors, it is important to be able to test for somatic changes in routinely available tumour samples. Such samples are typically formalin-fixed paraffin-embedded (FFPE) tissue, where the extracted DNA tends to be highly fragmented and of limited quantity, making analysis of large genes such as BRCA1 and BRCA2 challenging. This is made more difficult as somatic changes may be evident in only part of the sample, due to the presence of normal tissue. Methods We examined the feasibility of analysing DNA extracted from FFPE ovarian and breast tumour tissue to identify significant DNA variants in BRCA1/ BRCA2 using next generation sequencing methods that were sensitive enough to detect low level mutations, multiplexed to reduce the amount of DNA required and had short amplicon design. The utility of two GeneRead DNAseq Targeted Exon Enrichment Panels with different designs targeting only BRCA1/2 exons, and the Ion AmpliSeq BRCA community panel, followed by library preparation and adaptor ligation using the TruSeq DNA PCR-Free HT Sample Preparation Kit and NGS analysis on the MiSeq were investigated. Results Using the GeneRead method, we successfully analysed over 76% of samples, with >95% coverage of BRCA1/2 coding regions and a mean average read depth of >1000-fold. All mutations identified were confirmed where possible by Sanger sequencing or replication to eliminate the risk of false positive results due to artefacts within FFPE material. Admixture experiments demonstrated that BRCA1/2 variants could be detected if present in >10% of the sample. A sample subset was evaluated using the Ion AmpliSeq BRCA panel, achieving >99% coverage and sufficient read depth for a proportion of the samples. Conclusions Detection of BRCA1/2 variants in fixed tissue is feasible, and could be performed prospectively to facilitate optimum treatment decisions for ovarian or breast cancer patients. Electronic supplementary material The online version of this article (doi:10.1186/s12907-015-0004-6) contains supplementary material, which is available to authorized users.

Background Neurofibromatosis type 2 (NF2) is an autosomal dominant condition with high spontaneous mutation rate which predisposes to the development of multiple nerve sheath tumours (schwannomas), meningiomas and ependymoma. The cardinal feature and main diagnostic criterion for the diagnosis of NF2 remains the development of bilateral vestibular schwannoma (BVS). With increasing use of MRI screening the possibility of a ‘chance’ diagnosis of BVS has been mooted with a potential frequency of one in two million people in their lifetime. Until now, however, no evidence for such an event has been published. We aimed to demonstrate that chance occurrence can occur and to estimate its frequency among those with just BVS late in life. Methods Two vestibular schwannomas from the same patient were DNA sequenced and underwent loss of heterozygosity analysis. Results We show that a man who developed BVS, at ages 52 and 67 years developed these tumours sporadically by demonstrating that there were no molecular events in common between the two tumours. Furthermore from a database of over 1200 patients with NF2, we have estimated that ∼25% of cases of BVS over 50 years and 50% over 70 years of age where no other features of NF2 are present represent a chance occurrence rather than due to an underlying mosaic or constitutional NF2 mutation. Conclusions Patients presenting with BVS later in life should be appraised of the potential likelihood they may not have NF2 and the resultant further reduction in risks of transmission to offspring.

Background: In the past 15 years, clinical laboratory science has been transformed by the use of technologies that cross the traditional boundaries between laboratory disciplines. However, during this period, issues of quality have not always been given adequate attention. The European Molecular Genetics Quality Network (EMQN) has developed a novel external quality assessment scheme for evaluation of DNA sequencing. We report the results of an international survey of the quality of DNA sequencing among 64 laboratories from 21 countries. Methods: Current practice for DNA sequence analysis was established by use of an online questionnaire. Participating laboratories were provided with 4 DNA samples of validated genotype. Evaluation of the results included assessing the quality of sequence data, variant genotypes, and mutation nomenclature. To accommodate variations in mutation nomenclature, variants indicated by participants were scored for compliance with 3 acceptable marking schemes. Results: A total of 346 genotypes were analyzed. Of these, 19 (5%) genotyping errors were made. Of these, 10 (53%) were false-negative and 9 (47%) were false-positive results. A further 27 (8%) errors were made in naming mutations. Results were analyzed for 3 indicators of data quality: PHRED quality scores, Quality Read Length, and Quality Read Overlap. Most laboratories produced results of acceptable diagnostic quality as judged by these indicators. The results were used to calculate a consensus benchmark for DNA sequencing against which individual laboratories could rank their performance. Conclusions: We propose that the consensus benchmark can be used as a baseline against which the aggregate and individual laboratory standard of DNA sequencing may be tracked from year to year.

Schwannomatosis is characterized by multiple peripheral and cranial nerve schwannomas that occur in the absence of bilateral 8th cranial nerve schwannomas. The latter is the main diagnostic criterion of neurofibromatosis type 2 (NF2), which is a related but distinct disorder. The genetic factors underlying the differences between schwannomatosis and NF2 are poorly understood, although available evidence implicates chromosome 22 as the primary location of the gene(s) of interest. To investigate this, we comprehensively profiled the DNA copy number in samples from sporadic and familial schwannomatosis, NF2, and a large cohort of normal controls. Using a tiling-path chromosome 22 genomic array, we identified two candidate regions of copy number variation, which were further characterized by a PCR-based array with higher resolution. The latter approach allows the detection of minute alterations in total genomic DNA, with as little as 1.5 kb per measurement point of nonredundant sequence on the array. In DNA derived from peripheral blood from a schwannomatosis patient and a sporadic schwannoma sample, we detected rearrangements of the immunoglobulin lambda (IGL) locus, which is unlikely to be due to a B-cell specific somatic recombination of IGL. Analysis of normal controls indicated that these IGL rearrangements were restricted to schwannomatosis/schwannoma samples. In the second candidate region spanning GSTT1 and CABIN1 genes, we observed a frequent copy number polymorphism at the GSTT1 locus. We further describe missense mutations in the CABIN1 gene that are specific to samples from schwannomatosis and NF2 and make this gene a plausible candidate for contributing to the pathogenesis of these disorders.

Objective: To screen for NF2 mutations in people with meningiomas. Methods: Lymphocyte or tumour DNA was analysed from 46 individuals from 36 families who presented with a meningioma at age ⩽15 years without vestibular schwannoma (VS), or who had multiple meningiomas in adulthood before the diagnosis of VS. Results: Eight of 13 people with meningioma and other features of neurofibromatosis 2 (NF2) had an identified constitutional NF2 mutation in blood DNA, but none of the other subjects had identified constitutional NF2 mutations. Conclusions: Constitutional NF2 mutations are the most likely cause of meningioma in children and in people with a meningioma plus other non-VS features of NF2. Mosaic NF2 may be the cause of about 8% of multiple meningiomas in sporadic adult cases, but there are other causes in the majority of other such patients and in multiple meningioma in families.

Neurofibromatosis 2 (NF2) is an autosomal dominant disease that is characterized by tumors on the vestibular branch of the VIII cranial nerve, but other types of nervous system tumors usually occur as well. Genotype-phenotype correlations are well documented for overall NF2 disease severity but have not been definitively evaluated for specific types of non–VIII nerve tumors. We evaluated genotype-phenotype correlations for various types of non–VIII nerve tumors in 406 patients from the population-based United Kingdom NF2 registry, using regression models with the additional covariates of current age and type of treatment center (specialty or nonspecialty). The models also permitted consideration of intrafamilial correlation. We found statistically significant genotype-phenotype correlations for intracranial meningiomas, spinal tumors, and peripheral nerve tumors. People with constitutional NF2 missense mutations, splice-site mutations, large deletions, or somatic mosaicism had significantly fewer tumors than did people with constitutional nonsense or frameshift NF2 mutations. In addition, there were significant intrafamilial correlations for intracranial meningiomas and spinal tumors, after adjustment for the type of constitutional NF2 mutation. The type of constitutional NF2 mutation is an important determinant of the number of NF2-associated intracranial meningiomas, spinal tumors, and peripheral nerve tumors.

Neurofibromatosis 2 (NF2), an autosomal dominant disorder that is characterised by tumours of cells of neural crest origin, is caused by inactivating mutations of the NF2 gene on chromosome 22q12.1,2 Bilateral vestibular schwannomas are the most frequent manifestation of the disease, but other central and peripheral nervous system schwannomas, cerebral meningiomas, and ocular abnormalities are also common.3–5 The birth incidence of NF2 is 1 in 33 000 to 1 in 40 000.6 Constitutional NF2 mutations have been found in 30-60% of NF2 patients, and genotype-phenotype correlations have been substantiated.7–12 Half of NF2 patients are new mutations,6 in whom constitutional NF2 mutations can occur either in parental germline cells (prezygotic) or in cells after fertilisation (postzygotic). Postzygotic mutations can result in mosaicism, defined as the presence of a mutation, deletion, or chromosomal abnormality in a group or population of cells.13 Owing to the rarity of NF2, most previous reports of mosaicism in NF2 have been limited in scope,14–16 although a recent study estimated that 25% of NF2 patients with new mutations are mosaic.17 In this study, we describe mutational analysis of 27 mosaic cases of NF2 and the results of genetic testing in their children. NF2 patients were identified through the United Kingdom NF2 registry in the Department of Medical Genetics, St Mary’s Hospital, Manchester. Patients are ascertained by contacting neurosurgeons, otolaryngologists, neurologists, paediatricians, dermatologists, and geneticists throughout the United Kingdom, augmented in the North West Region by the Regional Cancer Registry. There were 297 people from unrelated families who were screened for constitutional NF2 mutations; 256 had bilateral vestibular schwannomas and 41 met other Manchester clinical diagnostic criteria for NF2.3,18 Of these 297 families, 68 families had affected patients in more than one generation, but the …

Background: Four sets of clinical diagnostic criteria for neurofibromatosis 2 (NF2) have been developed by groups of expert clinicians, but sensitivity has never been formally assessed. The sets of criteria differ for people without bilateral vestibular schwannomas, which are pathognomonic for NF2. Objective: To empirically evaluate the four existing sets of clinical diagnostic criteria for NF2. Methods: The study was based on 163 of 403 people in the United Kingdom NF2 registry (41%) who presented without bilateral vestibular schwannomas. The authors applied the sets of criteria to each person at initial assessment and at the most recent clinical evaluation (mean ± SE length of follow-up, 13 ± 1 years). Results: In people with “definite NF2” and a negative family history of NF2, the 1987 US NIH and 1991 NIH criteria each identify 78% of people at the most recent clinical evaluation but 0% at initial assessment. The National Neurofibromatosis Foundation (NNFF) criteria and the Manchester criteria each identify higher proportions at both time points (NNFF criteria, 91% and 10%; Manchester criteria, 93% and 14%), but the proportions at initial assessment are still low. Conclusions: None of the existing sets of criteria are adequate at initial assessment for diagnosing people who present without bilateral vestibular schwannomas as having NF2, particularly people with a negative family history of NF2. The authors recommend that a single, revised set of diagnostic criteria be devised to replace all of the existing sets of criteria.

Measuring correlation in clinical traits among relatives is important to our understanding of the causes of variable expressivity in Mendelian diseases. Random effects models are widely used to estimate intrafamilial correlations, but such models have limitations. We incorporated survival techniques into a random effects model so that it can be used to estimate intrafamilial correlations in continuous variables with right censoring, such as age at onset. We also describe a negative-binomial gamma mixture model to determine intrafamilial correlations of discrete (e.g., count) data. We demonstrate the utility of these methods by analyzing intrafamilial correlations among patients with neurofibromatosis 2 (NF2), an autosomal-dominant disease caused by mutations of the NF2 tumor-suppressor gene. We estimated intrafamilial correlations in age at first symptom of NF2, age at onset of hearing loss, and number of intracranial meningiomas in 390 NF2 nonprobands from 153 unrelated families. A significant intrafamilial correlation was observed for each of the three features: age at onset (0.35; 95% confidence interval (CI) 0.23-0.47), age at onset of hearing loss (0.51; 95% CI, 0.35-0.64), and number of meninginomas (0.29; 95% CI, 0.15-0.43). Significant correlations were also observed for age at first symptom within NF2 families with truncating mutations (0.41; 95% CI, 0.06-0.68) or splice-site mutations (0.29; 95% CI, 0.03-0.51), for age at onset of hearing loss within families with missense mutations (0.67; 95% CI, 0.18-0.89), and for number of meningiomas within families with splice-site mutations (0.39; 95% CI, 0.13-0.66). Our findings are consistent with effects of both allelic and nonallelic familial factors on the clinical variability of NF2.

To evaluate clinical and molecular predictors of the risk of mortality in people with neurofibromatosis 2 (NF2), we analyzed the mortality experience of 368 patients from 261 families in the United Kingdom NF2 registry, using the Cox proportional-hazards model and the jackknife method. Age at diagnosis, intracranial meningiomas, and type of treatment center were informative predictors of the risk of mortality. In Cox models, the relative risk of mortality increased 1.13-fold per year decrease in age at diagnosis (95% confidence interval [CI] 1.08-1.18) and was 2.51-fold greater in people with meningiomas compared with those without meningiomas (95% CI 1.38-4.57). The relative risk of mortality in patients treated at specialty centers was 0.34 compared with those treated at nonspecialty centers (95% CI 0.12-0.98). In a separate model, the relative risk of mortality in people with constitutional NF2 missense mutations was very low compared with those with other types of mutations (nonsense or frameshift mutations, splice-site mutations, and large deletions), but the CI could not be well quantified because there was only one death among people with missense mutations. We conclude that age at diagnosis, the strongest single predictor of the risk of mortality, is a useful index for patient counseling and clinical management (as are intracranial meningiomas). To ensure optimal care, we recommend that people with NF2 be referred to specialty treatment centers.

We recently reported SMARCE1 mutations as a cause of spinal clear cell meningiomas. Here, we have identified five further cases with non-NF2 spinal meningiomas and six with non-NF2 cranial meningiomas. Three of the spinal cases and three of the cranial cases were clear cell tumours. We screened them for SMARCE1 mutations and investigated copy number changes in all point mutation-negative samples. We identified two novel mutations in individuals with spinal clear cell meningiomas and three mutations in individuals with cranial clear cell meningiomas. Copy number analysis identified a large deletion of the 5' end of SMARCE1 in two unrelated probands with spinal clear cell meningiomas. Testing of affected and unaffected relatives of one of these individuals identified the same deletion in two affected female siblings and their unaffected father, providing further evidence of incomplete penetrance of meningioma disease in males. In addition, we found loss of SMARCE1 protein in three of 10 paraffin-embedded cranial clear cell meningiomas. Together, these results demonstrate that loss of SMARCE1 is relevant to cranial as well as spinal meningiomas. Our study broadens the spectrum of mutations in the SMARCE1 gene and expands the phenotype to include cranial clear cell meningiomas.

Static correlation energies (Estat) are calculated in a range of basis sets for a chemically diverse collection of atoms and molecules. The reliability of a basis set in capturing Estat is assessed according to the following: mean and maximum absolute deviations from near-exact Estat estimates, monotonic convergence to the complete basis set limit, and ability to capture Estat accurately independent of changes in geometry, molecular size, and electronic configuration. Within the polarization and correlation-consistent basis set series, triple-ζ basis sets are the smallest that can reliably capture Estat. The cc-pVTZ basis set performs particularly well, recovering Estat to chemical accuracy for all atoms and molecules in our data set. A series of customized basis sets are constructed by stripping polarization functions from, and swapping polarization functions among, existing basis sets. Basis sets without polarization functions are incapable of accurately recovering Estat. Basis sets with a near-complete set of s, p, and d functions can approach chemical accuracy in maximum absolute error. However, this may be achieved at lower computational cost by using a well balanced triple-ζ basis set including f functions, along with a smaller number of s, p, and d functions. Recommended basis sets for calculating Estat with increasing accuracy at increasing computational cost are 6-311G(2d,2p), cc-pVTZ, and cc-pVQZ stripped of g functions.

The spectrum of CFTR mutations in three South African populations is presented. To date. a total of 192 white patients (384 chromosomes) with confirmed CF have been tested. deltaF508 accounts for 76% of the CF chromosomes in this group, with 3272-26A-->G, 394delTT and G542X occurring at the following frequencies: 4, 3.6 and 1.3%, respectively. A further 11 mutations account for 6% of CF chromosomes. A total of 91% of the CF-causing mutations can now be detected in the South African white population. Haplotype analysis suggests a founder effect in South Africans of European origin for the two common CFTR mutations, 3272-26A-->G and 394delTT. The diagnosis of CF has been confirmed in 14 coloured and 12 black CF patients. In the coloured population, both the deltaF508 and 3120 + 1G-->A mutations occur at appreciable frequencies of 43 and 29%, respectively. In the black population, the most common CF-causing mutation, the 3120 + 1G-->A mutation, occurs at an estimated frequency of 46%. Four other mutations have been detected, resulting in the identification of a total of 62.5% of mutations in this population.

Genetic testing of an Irish kindred identified an exonic nucleotide substitution c.1664T>C (p.Leu555Pro) in the MLH1 mismatch repair (MMR) gene. This previously unreported variant is classified as a “variant of uncertain significance” (VUS). Immunohistochemical (IHC) analysis and microsatellite instability (MSI) studies, genetic testing, a literature and online MMR mutation database review, in silico phenotype prediction tools, and an in vitro MMR activity assay were used to study the clinical significance of this variant. The MLH1 c.1664T>C (p.Leu555Pro) VUS co-segregated with three cases of classic Lynch syndrome-associated malignancies over two generations, with consistent loss of MLH1 and PMS2 protein expression on IHC, and evidence of the MSI-High mutator phenotype. The leucine at position 555 is well conserved across a number of species, and this novel variant has not been reported as a normal polymorphism in the general population. In silico and in vitro analyses suggest that this variant may have a deleterious effect on the MLH1 protein and abrogate MMR activity. Evidence from clinical, histological, immunohistochemical, and molecular genetic data suggests that MLH1 c.1664T>C (p.Leu555Pro) is likely to be the pathogenic cause of Lynch syndrome in this family.

Editor—We have previously reported on a large clinical study of patients with neurofibromatosis 2 (NF2)1and described the usefulness of audiological screening in early detection of VS in NF2.2 Although DNA diagnosis is possible by linkage analysis and by detecting the NF2 family specific mutation, this is not possible in all cases. Only 40% of sporadic patients (new mutations) have an identifiable NF2 mutation3 and as 50% of NF2 patients have no family history,4 up to 30% of those at risk of NF2 will not be able to have a DNA presymptomatic test. While it is possible to calculate residual risks of NF2 from age at onset of symptoms curves,5 a more useful measure would be the level of risk reduction from a normal cranial MRI scan. A database recording information on affected subjects with NF2 was set up in Manchester in 1989. Patients were actively sought across the UK from paediatricians, neurosurgeons, ENT surgeons, geneticists, and dermatologists. We have used this database to determine the age at which MRI detects asymptomatic tumours in subjects with NF2 and the value of audiological screening. We have also created life curves for risk reduction in subjects at 50% risk of NF2. Patient details from hospital notes and proformas have been entered into a database at St Mary's Hospital Manchester since 1989. A total of 344 patients fulfilling our published criteria for NF2 are recorded (table 1).1 3 5 Information on all known tumours, age at presentation and diagnosis, and many different symptoms are included. We have analysed details of patients who have been detected as having VS on a screening MRI scan without any known central nervous system pathology, signs, or symptoms. It has been our practice to carry out a baseline MRI …

Many mutation scanning techniques are capable of locating mutations in DNA fragments much larger than the average exon. We have developed a system called Meta-PCR that can maximize the length of sequence scanned by these techniques, improving their productivity and realizing their full potential. Meta-PCR is a simple, versatile, and powerful method for generating chimeric DNA molecules. Currently, up to five PCR amplifiable fragments can be combined to form a single linear amplimer. The Meta-PCR reaction is self-assembling and takes place in two coupled stages carried out in a single reaction vessel. The order of fragments is reproducible and determined by primer design. We have developed two Meta-PCR assays, one comprising exons 6-10 of the Neurofibromatosis type 2 (NF2) gene and the second exons 8-12 of the human mismatch repair gene, hMLH1. We verified by direct sequencing that the order and sequence of the component exons in the Meta-PCR products is as predicted. Meta-PCR products from seven previously ascertained heterozygotes for NF2 mutations were directly sequenced. All seven mutations were clearly visible as mixed bases at the expected nucleotide, confirming that Meta-PCR faithfully reproduces the original sample genotype. We have evaluated the downstream use of the NF2 Meta-PCR products in fluorescent solid-phase chemical cleavage of mismatches (CCM). Meta-PCR products from eleven NF2 mutant heterozygotes were screened retrospectively for piperidine cleavage after hydroxylamine or potassium permanganate modification of mismatched bases. Ten of the 11 mutants were detected by visible cleavage. One mutation predicted to be cleaved after potassium permanganate modification was not detected. However, we were able to attribute this false negative to a failure in the CCM method. Meta-PCR is likely to be useful to clinical molecular diagnostic laboratories, helping them to fulfill demand for rapid and accurate screening for point mutations in large multi-exon genes.

Blood samples from 125 families with classic type 2 neurofibromatosis with bilateral vestibular schwannomas were analyzed for mutations in the NF2 gene. Causative mutations were identified in 52 families. In five families, the first affected individual in the family (the index case) was a mosaic for a disease-causing mutation. Only one of nine children from the three mosaic cases with children are affected. Four of these nine children inherited the allele associated with the disease-causing mutation yet did not inherit the mutation. NF2 mutations were identified in only 27/79 (34%) of sporadic cases, compared with 25/46 (54%) of familial cases (P

Blood samples from 125 unrelated families with classical type 2 neurofibromatosis (NF2) with bilateral vestibular schwannomas have been analysed for mutations in the NF2 gene. A further 17 families fulfilling modified criteria for NF2 have also been analysed. Causative mutations have been identified in 54 (43%) classical families and six (35%) of those fulfilling modified criteria. Forty-two cases from 38 families with truncating mutations had an average age at onset of symptoms of 19 years and diagnosis at 22.4 years. Fifty-one cases from 16 families with splice site mutations (15 from six), missense mutations (18 from six), and large deletions (18 from five) had an average age of onset of 27.8 years and at diagnosis of 33.4 years. Subjects with truncating mutations were significantly more likely to have symptoms before 20 years of age (p

TPS11614Background: Molecular stratification of patients to early phase trials of targeted therapies is a rational approach to drug development to help maximise benefit for individuals and provide ...

Mutations of the SMARCB1 gene have been implicated in several human tumour predisposing syndromes. They have recently been identified as an underlying cause of the tumour suppressor syndrome schwannomatosis. There is a much higher rate of mutation detection in familial disease than in sporadic disease. We have carried out extensive genetic testing on a cohort of familial and sporadic patients who fulfilled clinical diagnostic criteria for schwannomatosis. In our current cohort, we identified novel mutations within the SMARCB1 gene and detected several mutations that have been previously identified in other schwannomatosis cohorts. Of the schwannomatosis screens reported to date, including our current dataset, SMARCB1 mutations have been found in 45 % of familial probands and 7 % of sporadic patients. The exon 1 mutation, c.41C >A, and the 3′ untranslated region mutation, c.*82C >T, are the most common changes reported in schwannomatosis disease so far, indicating mutation hotspots at both 5′ and 3′ portions of the gene. SMARCB1 mutations are found in a significant proportion of schwannomatosis patients, but there remains the possibility that further causative genes remain to be found.

Background Meningiomas have been reported to occur in approximately 50% of neurofibromatosis type 2 (NF2) patients. The NF2 gene is commonly biallelically inactivated in both schwannomas and meningiomas. The spectrum of NF2 mutations consists mainly of truncating (nonsense and frameshift) mutations. A smaller number of patients have missense mutations, which are associated with a milder disease phenotype. Methods This study analysed the cumulative incidence and gender effects as well as the genotype–phenotype correlation between the position of the NF2 mutation and the occurrence of cranial meningiomas in a cohort of 411 NF2 patients with proven NF2 mutations. Results and Conclusion Patients with mutations in exon 14 or 15 were least likely to develop meningiomas. Cumulative risk of cranial meningioma to age 50 years was 70% for exons 1–3, 81% for exons 4–6, 49% for exons 7–9, 56% for exons 10–13, and 28% for exons 14–15. In the cohort of 411 patients, no overall gender bias was found for occurrence of meningioma in NF2 disease. Cumulative incidence of meningioma was close to 80% by 70 years of age for both males and females, but incidence by age 20 years was slightly increased in males (male 25%, female 18%; p=0.023). Conversely, an increased risk of meningiomas in women with mosaic NF2 disease was also found.

Background Translocations of the Mixed Lineage Leukemia (MLL) gene occur in a subset (5%) of acute myeloid leukemias (AML), and in mixed phenotype acute leukemias in infancy - a disease with extremely poor prognosis. Animal model systems show that MLL gain of function mutations may contribute to leukemogenesis. Wild-type (wt) MLL possesses histone methyltransferase activity and functions at the level of chromatin organization by affecting the expression of specific target genes. While numerous MLL fusion proteins exert a diverse array of functions, they ultimately serve to induce transcription of specific genes. Hence, acute lymphoblastic leukemias (ALL) with MLL mutations (MLL mu) exhibit characteristic gene expression profiles including high-level expression of HOXA cluster genes. Here, we aimed to relate MLL mutational status and tumor suppressor gene (TSG) methylation/expression in acute leukemia cell lines. Results Using MS-MLPA (methylation-specific multiplex ligation-dependent probe amplification assay), methylation of 24 different TSG was analyzed in 28 MLL mu and MLL wt acute leukemia cell lines. On average, 1.8/24 TSG were methylated in MLL mu AML cells, while 6.2/24 TSG were methylated in MLL wt AML cells. Hypomethylation and expression of the TSG BEX2, IGSF4 and TIMP3 turned out to be characteristic of MLL mu AML cell lines. MLL wt AML cell lines displayed hypermethylated TSG promoters resulting in transcriptional silencing. Demethylating agents and inhibitors of histone deacetylases restored expression of BEX2, IGSF4 and TIMP3, confirming epigenetic silencing of these genes in MLL wt cells. The positive correlation between MLL translocation, TSG hypomethylation and expression suggested that MLL fusion proteins were responsible for dysregulation of TSG expression in MLL mu cells. This concept was supported by our observation that Bex2 mRNA levels in MLL-ENL transgenic mouse cell lines required expression of the MLL fusion gene. Conclusion These results suggest that the conspicuous expression of the TSG BEX2, IGSF4 and TIMP3 in MLL mu AML cell lines is the consequence of altered epigenetic properties of MLL fusion proteins.

The LMNA gene was sequenced in 98 Czech patients from 94 unrelated families with early-onset axonal Charcot–Marie–Tooth (CMT) disease consistent with both autosomal recessive inheritance and sporadic cases. Biallelic pathogenic mutations were not found in any patient in this group. One patient carried the c.1870C>T mutation that is predicted to result in the amino-acid substitution, p. Arg624Cys, on one allele, but the second causative mutation was not detected. LMNA mutation is not likely to be associated with the disease in this family. To exclude larger deletions/duplications in the LMNA gene not detectable by sequencing, 48 patients from this group were also analyzed with multiplex ligation-dependent probe amplification. No rearrangements in the LMNA gene were detected. We conclude that mutations in the LMNA gene are absent from a large group of Czech patients with axonal autosomal recessive CMT disease. Consequently, LMNA mutation screening does not seem to be relevant for axonal CMT DNA diagnostics. A similar situation may apply to other European populations.

The past 10 years have seen an improvement in sequence data quality due to the introduction of capillary sequencers and new sequencing chemistries. In parallel, new software programs for automated mutation detection have been developed. We evaluated the sensitivity of semiautomated unidirectional sequence analysis for the detection of heterozygous base substitutions using the Mutation Surveyor software package.Detection rates for heterozygous base substitutions in 29 genes by automated and visual inspection were compared. Examples of heterozygous bases not detected in one direction during bidirectional analysis were also sought through a national survey of United Kingdom (UK) genetics laboratories. Sequence quality was assessed in a consecutive cohort of 50 patients for whom the 39 exons of the ABCC8 gene had been sequenced in one direction.A total of 701 different heterozygous base substitutions were detected by the software with no false negatives (sensitivityor=99.57%). Four examples of heterozygous bases missed in one direction during bidirectional analysis were reported. Two were detected using unidirectional analysis settings, and the other two bases had low-quality scores. Of the 1950 amplicons examined, 97.2% had a quality scoreor=30 and an average PHRED-like scoreor=50 for the defined region of interest, and 98.1% of the 323,650 bases had a PHRED score40.We found no evidence to support a requirement for bidirectional sequencing. Semiautomated analysis of good quality unidirectional sequence data has high sensitivity and is suitable for heterozygote mutation scanning in clinical diagnostic laboratories. Further work is required to determine minimum quality parameters for semiautomated analysis.

64 families with a history of male breast cancer aged 60 or less or with a family history of male and female breast cancer were screened for the presence of BRCA1 and BRCA2 mutations. Seventeen pathogenic BRCA2 and four BRCA1 mutations were identified (34%) in samples from an affected family member. All but one of the mutations segregated with disease where samples were available and pedigree structure permitted. Despite high sensitivity of mutation testing only 64% of families fulfilling BCLC criteria had an identifiable pathogenic mutation. It is possible that at least some of these families may have mutations in other genes, although we found no involvement of CHEK2 1100delC.

A two generation family with neurofibromatosis type 2 (NF2) is presented in which a family member requested presymptomatic molecular diagnosis. Since the consultand's mother had clinically well defined NF2, he was quoted to be at 50% risk of carrying an NF2 mutation. Mutation screening in the mother did not show the causative mutation and, consequently, presymptomatic testing was based on linkage analysis. This showed that the consultand carried the high risk chromosome 22. Subsequent mutation screening of his clinically affected sister showed a nonsense mutation, R262X in exon 8 of the NF2 gene. The mother turned out to be a mosaic for R262X; the son had not inherited the mutation. Mosaicism may be a common mechanism in NF2 and other autosomal dominant diseases with a high new mutation rate. This may be one explanation for a difference in expression in generations. Caution has to be exercised when giving results based on linkage tests which imply a very high risk to people in the second generation.

Methods: DNA samples from affected subjects from 422 non-Jewish families with a history of breast and/or ovarian cancer were screened for BRCA1 mutations and a subset of 318 was screened for BRCA2 by whole gene screening techniques. Using a combination of results from screening and the family history of mutation negative and positive kindreds, a simple scoring system (Manchester scoring system) was devised to predict pathogenic mutations and particularly to discriminate at the 10% likelihood level. A second separate dataset of 192 samples was subsequently used to test the model's predictive value. This was further validated on a third set of 258 samples and compared against existing models.

Neurofibromatosis 2 (NF2) patients with constitutional splice site NF2 mutations have greater variability in disease severity than NF2 patients with other types of mutations; the cause of this variability is unknown. We evaluated genotype-phenotype correlations, with particular focus on the location of splice site mutations, using mutation and clinical information on 831 patients from 528 NF2 families with identified constitutional NF2 mutations. The clinical characteristics examined were age at onset of symptoms of NF2 and number of intracranial meningiomas, which are the primary indices of the severity of NF2. Two regression models were used to analyse genotype-phenotype correlations. People with splice site mutations in exons 1–5 had more severe disease than those with splice site mutations in exons 11–15. This result is compatible with studies showing that exons 2 and 3 are required for self-association of the amino terminal of the NF2 protein in vitro, and that deletions of exons 2 and 3 in transgenic and knockout mouse models of NF2 cause a high prevalence of Schwann cell derived tumours.

Speed dependent automatic zooming (SDAZ) is a promising refinement to scrolling in which documents are automatically zoomed-out as the scroll rate increases. By automatically zooming, the visual flow rate is reduced enabling rapid scrolling without motion blur. In order to aid SDAZ calibration we theoretically and empirically scrutinise human factors of the speed/zoom relationship. We then compare user performance with four alternative text-document scrolling systems, two of which employ automatic zooming. One of these systems, which we term 'DDAZ', is based on van Wijk and Nuij's recent and important theory that calculates optimal pan/zoom paths between known locations in 2D space. van Wijk and Nuij suggested that their theory could be applied to scrolling, but did not implement or test their formulaic suggestions. Participants in our evaluation (n=27) completed scrolling tasks most rapidly when using SDAZ, followed by DDAZ, normal scrollbars, and traditional rate-based scrolling. Workload assessments and preferences strongly favoured SDAZ. We finish by examining issues for consideration in commercial deployments.

We describe the development and implementation of a neurofibromatosis type 2 (NF2) mutation scanning service based on novel techniques. All 17 exons of the NF2 gene are amplified in four polymerase chain reaction (PCR) reactions, using the meta-PCR technique to link the NF2 exons into chimeric concatamers. The meta-PCR products are then scanned for point mutations by direct sequencing. A four-exon dosage assay is used to test for large deletion/duplication mutations. In certain cases when tumour studies are necessary, these techniques are also combined with loss of heterozygosity analysis with three highly polymorphic microsatellite markers located within or close to the NF2 gene. Over a period of 2 years, we have applied these techniques in a service setting to the analysis of 271 patient samples (245 lymphocyte DNA; 26 schwannoma DNA). Meta-PCR and sequencing identified 90 point mutations in the 271 blood and tumor samples, 48 of which have not been reported previously. Dosage analysis identified large deletions in 12 of the lymphocyte DNA samples. In addition, over 84% of mutations were identified in 23 schwannoma DNA samples in which complete analysis was possible. Adoption of this novel strategy has increased the overall mutation detection rate in familial NF2 cases to 88% and sporadic NF2 cases to 59%. It has also allowed us to decrease our reporting turnaround times, and because of a low overall failure rate, permitted the running of an efficient and cost-effective service.

Neurofibromatosis type 2 (NF2) is an autosomal dominant condition characterised by vestibular schwannomas, schwannomas of other cranial nerves, meningiomas, and other low grade brain malignancies.1 The severity of NF2 is variable, with some patients having early onset disease and more rapidly growing tumours that occur in greater numbers. The NF2 gene is on chromosome 22q12.2,3 The protein product (termed merlin or schwannomin) is a cell cytoskeleton associating protein. Genotype−phenotype correlations have been demonstrated, with missense mutations and large deletions causing mild disease, and nonsense or frameshift mutations causing severe disease.4,5 The current mutation screening techniques of single strand conformation polymorphism analysis (SSCP), protein truncation test, and denaturing gradient gel electrophoresis detect 33–65% of mutations, although adding a deletion strategy increases the proportion to 80%.6 However, deletion testing and chromosome analysis are rarely reported in studies of NF2 mutations. In this study, we present five NF2 patients for whom chromosome analysis, usually following routine molecular screening, revealed the underlying genetic aberration. ### Case 1 A 20 year old female was referred with a large diffuse goitre commencing early in puberty, moderate learning difficulties, and bilateral sensorineural hearing loss, which resembles the phenotype of Pendred’s syndrome. She was born after a normal pregnancy and delivery. Her physical and mental development during early infancy was not remarkable, but developmental delay became apparent in the second year of life. Growth parameters were within the normal range. She began to walk at the age of 3 years. Six months later, audiometric tests showed no hearing impairment although chronic otitis media was diagnosed on one side. According to the parents, her communication had been improving with age, but her speech remained dysarthric and her articulation was very poor. In primary school, she had sub-average grades in all subjects. Her affect was good and …

Neurofibromatosis type 2 (NF2) is an autosomal-dominant disorder affecting about 1:33 000 newborns, mainly characterized by the development of tumors of the nervous system and ocular abnormalities. Around 85% of germline NF2 mutations are point mutations. Among them, ∼25% affect splicing and are associated with a variable disease severity. In the context of our NF2 Multidisciplinary Clinics, we have identified a patient fulfilling clinical criteria for the disease and exhibiting a severe phenotype. The patient carries a deep intronic mutation (g. 74409T>A, NG_009057.1) that produces the insertion of a cryptic exon of 167pb in the mature mRNA between exons 13 and 14, resulting in a truncated merlin protein (p.Pro482Profs*39). A mutation-specific antisense phosphorodiamidate morpholino oligomer was designed and used in vitro to effectively restore normal NF2 splicing in patient-derived primary fibroblasts. In addition, merlin protein levels were greatly recovered after morpholino treatment, decreasing patient's fibroblasts in vitro proliferation capacity and restoring cytoeskeleton organization. To our knowledge, this is the first NF2 case caused by a deep intronic mutation in which an in vitro antisense therapeutic approximation has been tested. These results open the possibility of using this approach in vivo for this type of mutation causing NF2.

Unstable trinucleotide repeats are a newly recognized class of disease mutation. Several major human single gene disorders are now attributed to expansions of these highly unstable sequences (1-4). Their molecular analysis is particularly challenging, since: 1. Accurate allele sizing is essential; 2. Polymerase chain reaction (PCR) amplification across the repeat is hampered by extreme guanme cytosme (GC) content and strong secondary structure, and 3. Size differences between normal and mutated alleles may be great, for instance, in fragile X they can range from 6 to over 1000 repeats.

We report two unusual patients with trisomy 18 mosaicism presenting with minor anomalies and failure to thrive in the first year of life. Chromosome analysis showed trisomy 18 in 30/30 peripheral blood lymphocytes in both children. Analysis of skin fibroblasts in the first child showed normal female chromosomes in 30/30 cells, and the fibroblast karyotype in the second child showed mosaicism for tetrasomy 18p, trisomy 18, and normal female chromosomes (karyotype 47,XX, +i(18)(p10)[47]/47,XX, +18[9] /46,XX[4]). Trisomy 18 commonly results from nondisjunction at maternal meiosis II (MII). Nondisjunction at maternal MII has also been postulated to be the initial step in the formation of tetrasomy 18p. In our second case, the additional chromosome 18 was the result of maternal nondisjunction at MII, consistent with this hypothesis. In the first case, nondisjunction at maternal meiosis I (MI) was responsible for the extra chromosome 18. © 2002 Wiley-Liss, Inc.

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.