Donatella Bianchessi

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.

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.

Non-coding RNAs (ncRNAs) are known to regulate gene expression at the transcriptional and post-transcriptional levels, chromatin remodeling, and signal transduction. The identification of different species of ncRNAs, microRNAs (miRNAs), circular RNAs (circRNAs), and long ncRNAs (lncRNAs)—and in some cases, their combined regulatory function on specific target genes—may help to elucidate their role in biological processes. NcRNAs’ deregulation has an impact on the impairment of physiological programs, driving cells in cancer development. We here carried out a review of literature concerning the implication of ncRNAs on tumor development in neurofibromatosis type 1 (NF1), an inherited tumor predisposition syndrome. A number of miRNAs and a lncRNA has been implicated in NF1-associated tumors, such as malignant peripheral nerve sheath tumors (MPNSTs) and astrocytoma, as well as in the pathognomonic neurofibromas. Some authors reported that the lncRNA ANRIL was deregulated in the blood of NF1 patients with plexiform neurofibromas (PNFs), even if its role should be further elucidated. We here provided original data concerning the association of a specific genotype about ANRIL rs2151280 with the presence of optic gliomas and a mild expression of the NF1 phenotype. We also detected the LOH of ANRIL in different tumors from NF1 patients, supporting the involvement of ANRIL in some NF1-associated tumors. Our results suggest that ANRIL rs2151280 may be a potential diagnostic and prognostic marker, addressing early diagnosis of optic glioma and predicting the phenotype severity in NF1 patients.

Neurofibromatosis type I (NF1) microdeletion syndrome, which is present in 4–11% of NF1 patients, is associated with a severe phenotype as it is caused by the deletion of NF1 and other genes in the 17q11.2 region. The variable expressivity of the disease makes it challenging to establish genotype–phenotype correlations, which also affects prognosis and counselling. We here describe a 3-year-old NF1 patient with an atypical deletion and a complex phenotype. The patient showed overgrowth, café au lait spots, inguinal freckling, and neurological abnormalities. The extent of the deletion was determined by means of array comparative genomic hybridisation, and its breakpoints were isolated by means of long-range polymerase chain reaction. Sequence analysis of the deletion junction fragment revealed the occurrence of an Alu-mediated recombination that led to the generation of a chimeric gene consisting of three exons of RNF135 and eleven exons of SUZ12. Interestingly, the deletion shares a common RNF135-centred region with another deletion described in a non-NF1 patient with overgrowth. In comparison with the normal RNF135 allele, the chimeric transcript was 350-fold over-expressed in peripheral blood, and the ADAP2 gene located upstream of RNF135 was also up-regulated. In line with this, the deletion causes the loss of a chromatin TD boundary, which entails the aberrant adoption of distal cis-acting regulatory elements. These findings suggest that RNF135 haploinsufficiency is related to overgrowth in patients with NF1 microdeletion syndrome and, for the first time, strongly indicate a position effect that warrants further genotype–phenotype correlation studies to investigate the possible existence of previously unknown pathogenic mechanisms.

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.

Although anti-VEGF therapy is widely used in high-grade gliomas, no predictor of response or toxicity has been reported yet. We investigated here the association of the functional single nucleotide polymorphism (SNP) rs2010963, located in the 5′ untranslated terminal region of the VEGFA gene, with survival, response to bevacizumab (BVZ) and vascular toxicity. The rs2010963 was genotyped by Taqman assay in blood DNA from 954 glioma patients with available survival data, including 225 glioblastoma (GBM) patients treated with BVZ. VEGFA plasma levels were assessed by ELISA in 87 patients before treatment. Thrombo-hemorragic adverse events were recorded during BVZ treatment or not, and in an independent population of 92 GBM patients treated with temozolomide. The CC genotype was associated with the occurrence of thrombo-hemorragic events (CC 25 versus CG 13.5 and GG 5.2 %; P = 0.0044) during BVZ. A similar but weaker and non significant trend was observed in patients not receiving BVZ. A CC genotype was associated with higher levels of plasma VEGFA at baseline (107.6 versus 57.50 pg/mL in heterozygotes (CG) and 52.75 pg/mL in GG patients, P = 0.035 and P = 0.028 respectively). The CC genotype tended to be associated to longer PFS when treated with BVZ (P = 0.05), but not when treated with the temozolomide treatment. Our data suggest that the rs2010963 genotype is associated with longer PFS, higher risk of vascular events in recurrent GBM especially treated with BVZ, and higher plasma VEGFA concentration. It may help to identify patients at risk of vascular adverse events during BVZ treatment.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder, which confers an increased risk of a wide range of cancers, and malignant tumors are the most common cause of death in individuals with NF1. Although in children with NF1, the most common neoplasms are optic nerve gliomas and brain tumors, an elevated risk of myeloid leukemia and an increased relative risk of acute lymphoblastic leukemia and non-Hodgkin lymphoma were reported. In adults with NF1, the relative risk of brain tumor is 100 times higher than in the general population. Cases of malignant lymphoma occurring in NF1 adult patients have been reported. However, the association between NF1 and lymphoproliferative diseases is still debated. We report a case of CNS primitive lymphoma in an adult patient who resulted positive for NF1 at genetic testing. At present, only one case of CNS lymphoma in an adult patient displaying clinical criteria for NF1 diagnosis has been reported.

Meningiomas are the most frequent intracranial tumors. Surgery can be curative, but recurrences are possible. We performed gene expression analyses and loss of heterozygosity (LOH) studies looking for new markers predicting the recurrence risk. We analyzed expression profiles of 23 meningiomas (10 grade I, 10 grade II, and 3 grade III) and validated the data using quantitative polymerase chain reaction (qPCR). We performed LOH analysis on 40 meningiomas, investigating chromosomal regions on 1p, 9p, 10q, 14q, and 22q. We found 233 and 268 probe sets to be significantly down- and upregulated, respectively, in grade II or III meningiomas. Genes downregulated in high-grade meningiomas were overrepresented on chromosomes 1, 6, 9, 10, and 14. Based on functional enrichment analysis, we selected LIM domain and actin binding 1 (LIMA1), tissue inhibitor of metalloproteinases 3 (TIMP3), cyclin-dependent kinases regulatory subunit 2 (CKS2), leptin receptor (LEPR), and baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5) for validation using qPCR and confirmed their differential expression in the two groups of tumors. We calculated ΔCt values of CKS2 and LEPR and found that their differential expression (C-L index) was significantly higher in grade I than in grade II or III meningiomas (p < .0001). Interestingly, the C-L index of nine grade I meningiomas from patients who relapsed in <5 years was significantly lower than in grade I meningiomas from patients who did not relapse. These findings indicate that the C-L index may be relevant to define the progression risk in meningioma patients, helping guide their clinical management. A prospective analysis on a larger number of cases is warranted.

Purpose: Recent data suggest that methylation of the DNA repair gene O⁶-methylguanine DNA methyltransferase (MGMT), by increasing the chemosensitivity of glioblastoma multiforme, is significantly associated with improved prognosis. Results in contradiction with these findings, however, are present in the literature and the clinical and genetic context framing MGMT methylation is poorly characterized. Experimental Design: To address these issues, we have investigated the MGMT methylation status, clinical and magnetic resonance imaging characteristics, and relevant genetic features (loss of heterozygosity on 17p and 19q, EGFR amplification, and p53 mutations) in a retrospective study on 86 patients affected by glioblastoma multiforme: 72 patients had a clinical history indicating de novo insurgence of the tumor and the remaining 14 were secondary glioblastoma multiforme. Results: MGMT methylation was detected by methylation-specific PCR in 41 of 86 cases (47.7%; Meth+). Progression-free survival and overall survival were significantly longer in Meth+ than in Meth− patients [10 versus 7 months (P = 0.003, log-rank test) and 18 versus 14 months (P = 0.0003, log-rank test), respectively]. Mixed-nodular enhancement at magnetic resonance imaging was significantly more frequent in Meth+ and secondary glioblastoma multiforme and ring enhancement in Meth− and primary glioblastoma multiforme (P < 0.005). MGMT methylation was more present in secondary glioblastoma multiforme (P = 0.006) and associated with loss of heterozygosity on 17p and/or 19q (P = 0.005). Conclusions: These observations suggest that MGMT methylation is part of a genetic signature of glioblastomas that developed from lower-grade gliomas.

Over the last decade, the knowledge on the molecular genetic background of gliomas has dramatically increased. This information provides the basis for the molecular target therapies and molecular tests serve to complement the subjective nature of histopathologic criteria and add useful data regarding response to treatments and prognosis. In particular, the use of loss of heterozygosity (LOH) and methylation specific polymerase chain reaction (PCR) (MSP) based testing of gliomas is already in place and used clinically in several centers. This paper provides a brief overview of these molecular genetic aberrations and discusses the clinical utility, as well as the advantages and disadvantages of such approach. Newly developed molecular techniques, such as LOH testing, fluorescence in situ hybridization (FISH), DNA sequencing and MSP, are currently being employed in assessment of gliomas in some laboratories. However, the clinical use of some markers and the context in which the information obtained should be used are still not entirely understood. Therefore, this paper will focus on validation and implementation of molecular testing in gliomas, with emphasis on LOH on chromosomes 1p, 19q, 17p and 10q and O⁶-methylguanine-DNA methyltransferase (MGMT) methylation status.