Simultaneous Detection of NF1, SPRED1, LZTR1, and NF2 Gene Mutations by Targeted NGS in an Italian Cohort of Suspected NF1 Patients.

Neurofibromatosis type 1 (NF1) displays overlapping phenotypes with other neurocutaneous diseases such as Legius Syndrome. Here, we present results obtained using a next generation sequencing (NGS) panel including NF1, NF2, SPRED1, SMARCB1, and LZTR1 genes on Ion Torrent. Together with NGS, the Multiplex Ligation-Dependent Probe Amplification Analysis (MLPA) method was performed to rule out large deletions/duplications in NF1 gene; we validated the MLPA/NGS approach using Sanger sequencing on DNA or RNA of both positive and negative samples. In our cohort, a pathogenic variant was found in 175 patients; the pathogenic variant was observed in NF1 gene in 168 cases. A SPRED1 pathogenic variant was also found in one child and in a one year old boy, both NF2 and LZTR1 pathogenic variants were observed; in addition, we identified five LZTR1 pathogenic variants in three children and two adults. Six NF1 pathogenic variants, that the NGS analysis failed to identify, were detected on RNA by Sanger. NGS allows the identification of novel mutations in five genes in the same sequencing run, permitting unambiguous recognition of disorders with overlapping phenotypes with NF1 and facilitating genetic counseling and a personalized follow-up.

Risk of Optic Pathway Glioma in Neurofibromatosis Type 1: No Evidence of Genotype-Phenotype Correlations in A Large Independent Cohort.

The occurrence of optic pathway gliomas (OPGs) in children with neurofibromatosis type 1 (NF1) still raises many questions regarding screening and surveillance because of the lack of robust prognostic factors. Recent studies of an overall cohort of 381 patients have suggested that the genotype may be the main determinant of the development of OPG, with the risk being higher in patients harbouring NF1 mutations in the 5' tertile and the cysteine/serine-rich domain. In an attempt to confirm this hypothesis, we used strict criteria to select a large independent cohort of 309 NF1 patients with defined constitutional NF1 mutations and appropriate brain images (255 directly enrolled and 54 as a result of a literature search). One hundred and thirty-two patients had OPG and 177 did not. The association of the position (tertiles and functional domains) and type of NF1 mutation with the development of OPG was analysed using the χ2 test and Fisher's exact probability test; odds ratios (ORs) with 95% confidence intervals were calculated, and Bonferroni's correction for multiple comparisons was applied; multiple logistic regression was also used to study genotype-phenotype associations further. Our findings show no significant correlation between the site/type of NF1 mutation and the risk of OPG, and thus do not support the hypothesis that certain constitutional mutations provide prognostic information in this regard. In addition, we combined our cohort with a previously described cohort of 381 patients for a total of 690 patients and statistically re-analysed the results. The re-analysis confirmed that there were no correlations between the site (tertile and domain) and the risk of OPG, thus further strengthening our conclusions.

126 novel mutations in Italian patients with neurofibromatosis type 1.

Genetic analysis of Neurofibromatosis type 1 (NF1) may facilitate the identification of patients in early phases of the disease. Here, we present an overview of our diagnostic research spanning the last 11 years, with a focus on the description of 225 NF1 mutations, 126 of which are novel, found in a series of 607 patients (513 unrelated) in Italy. Between 2003 and 2013, 443 unrelated patients were profiled by denaturing high pressure liquid chromatography (DHPLC) analysis of 60 amplicons derived from genomic NF1 DNA and subsequent sequencing of heterozygotic PCR products. In addition, a subset of patients was studied by multiplex ligation-dependent probe amplification (MLPA) to identify any duplications, large deletions or microdeletions present at the locus. Over the last year, 70 unrelated patients were investigated by MLPA and sequencing of 22 amplicons spanning the entire NF1 cDNA. Mutations were found in 70% of the 293 patients studied by DHPLC, thereby fulfilling the NIH criterion for the clinical diagnosis of NF1 (detection rate: 70%); furthermore, 87% of the patients studied by RNA sequencing were genetically characterized. Mutations were also found in 36 of the 159 patients not fulfilling the NIH clinical criteria. We confirmed a higher incidence of intellectual disability in patients harboring microdeletion type 1 and observed a correlation between a mild phenotype and the small deletion c.2970_2972delAAT or the missense alteration in amino acid residue 1809 (p.Arg1809Cys). These data support the use of RNA-based methods for genetic analysis and provide novel information for improving the management of symptoms in oligosymptomatic patients.

Dizziness in patients with recent episodes of benign paroxysmal positional vertigo: real otolithic dysfunction or mental stress?

OBJECTIVE: Our goal was to verify the existence of otolithic dysfunction or mental stress in patients with dizziness following an episode of benign paroxysmal positional vertigo (BPPV) that was treated and resolved. STUDY DESIGN: Prospective study. METHODS: Forty patients with BPPV were examined 2, 7, and 14 days after resolution. Based on residual symptoms reported during three follow-ups, they were classified as type A (with dizziness) or type B (without dizziness). During the first follow-up, they were asked to compile a self-rating anxiety scale (SAS), and we then determined subjective visual vertical (SVV). The determination of SVV was repeated during the subsequent follow-ups. For each patient, we also evaluated the rate of BPPV episodes that were recurrent and resistant to treatment and examined distribution by age and gender during the follow-ups. RESULTS: A comparison of type A (1.20 +/- 0.45) and type B (0.64 +/- 0.58) patients showed a significant difference in determining SVV only at the first follow-up (p = .002). Among type A patients, the rate of resistant BPPV was 75%, whereas the rate of recurrent BPPV was 100% at the third follow-up, during which the SAS revealed a significant increase (p = .005) among type A (52 +/- 3.74) versus type B (41.6 +/- 4.7) patients; the male to female ratio was 1:5 (type A) and 4:5 (type B), and the mean ages were, respectively, 56.4 +/- 4.98 and 43.6 +/- 10.2. CONCLUSIONS: Otolithic dysfunction explains only brief dizziness. The persistence of dizziness is correlated with mental stress that is affected by the duration and recurrence of BPPV, age, and gender.

Correlations between posturographic findings and symptoms in subjects with fractures of the condylar head of the mandible.

Our study examined the posture of 15 patients who had sustained a simple unilateral or bilateral fracture of the condylar head of the mandible as a result of sports or traffic accidents. Following preliminary testing of vestibular function, the patients underwent balance testing: Romberg test with eyes closed (EC), Romberg EC and bite test (ECBT), EC and head retroflexed (ECR). The study parameters were: surface (S) of the statokinesigram, stomatognathic influence index related to S (SSI), and postural oscillations on the frontal plane (X). In keeping with the literature, we felt that the following pattern in static balance suggested a posture destabilised by the stomatognathic system: SSI values of less than 60, reduction of S in the transition from EC to ECR, pathological increase of postural oscillations on the X plane. The study was completed by obtaining a list of new symptoms reported by the patients (altered bite, fullness, tinnitus, pain, loss of balance). The most significant patterns were observed in patients with vestibular dysfunctions and neck pain. It seems that a fracture of the condylar head can affect postural behaviour, although proprioceptive changes alone are not enough to cause true loss of balance and there must be concomitant vestibular dysfunction. The stabilometric pattern is not conditioned by the extent of the trauma or the related treatment. In terms of proprioceptive elements, the presence of muscle pain seems to point to cervical muscle tension as the main culprit in the onset of posttraumatic instability.