Neurofibromatosis type 1 (NF1) is a hereditary disorder caused by mutations in the NF1 gene. Detecting mutation in NF1 is hindered by the gene's large size, the lack of mutation hotspots, the presence of pseudogenes, and the wide variety of possible lesions. We developed a method for detecting germline mutations by combining an original RNA-based cDNA-PCR mutation detection method and denaturing high-performance liquid chromatography (DHPLC) with multiplex ligation-dependent probe amplification (MLPA). The protocol was validated in a cohort of 56 blood samples from NF1 patients who fulfilled NIH diagnostic criteria, identifying the germline mutation in 53 cases (95% sensitivity). The efficiency and reliability of this approach facilitated detection of different types of mutations, including single-base substitutions, deletions or insertions of one to several nucleotides, microdeletions, and changes in intragenic copy number. Because mutational screening for minor lesions was performed using cDNA and the characterization of mutated alleles was performed at both the RNA and genomic DNA level, the analysis provided insight into the nature of the different mutations and their effect on NF1 mRNA splicing. After validation, we implemented the protocol as a routine test. Here we present the overall unbiased spectrum of NF1 mutations identified in 93 patients in a cohort of 105. The results indicate that this protocol is a powerful new tool for the molecular diagnosis of NF1.

BackgroundNeurofibromatosis type 1 is a common autosomal dominant disorder with full penetrance and variable expression. The condition predisposes individuals to the development of malignant nervous system tumours, most frequently Malignant Peripheral Nerve Sheath Tumours (MPNSTs). Previous studies indicate that genetic factors other than mutations in NF1 may be responsible for the condition's variable expression.Case reportHere we present data from a pair of monozygotic twins affected by Neurofibromatosis type 1 resulting from a de novo mutation. Both twins developed a left sciatic plexiform neurofibroma that evolved into MPNST at a similar age and they also developed pulmonary metastasis at the same age. Other concordant traits between the twins were: macrocephaly, psychomotor delay, café-au-lait spots, cutaneous neurofibromas, retroperitoneal, pleural and paraspinal neurofibromas. The main discordant features observed were tibial pseudoarthrosis, pectus carinatum, osteoporosis and thymus hyperplasia.ConclusionsThis is the first report of monozygotic twins with Neurofibromatosis type 1 that develop MPNSTs, the localization and chronological evolution of which, and its metastasis, is concordant in both twins. These cases suggest that the events involved in the transformation of benign plexiform neurofibromas to MPNSTs in Neurofibromatosis type 1, follow a spatiotemporally programme that is influenced by heritable factors other than NF1 mutations.

Germline mutations in the SMARCB1 gene cause familial schwannomatosis, a condition characterized by the presence of multiple schwannomas, although mutations in SMARCB1 have also been associated with rhadboid tumor predisposition syndrome 1 (RTPS1). Both schwannomatosis and RTPS1 are autosomal dominant conditions that predispose individuals to develop distinct types of tumors. We clinically and genetically characterized two families with schwannomatosis associated with SMARCB1 mutations. Eight affected members of these families developed different numbers of schwannomas and/or meningiomas at distinct ages, evidence that meningiomas are variably expressed in this condition. We identified two germline mutations in SMARCB1 associated with the familial disease, c.233-1G>A and the novel c.207_208dupTA mutation, which both proved to affect the main SMARCB1 isoforms at the RNA level distinctly. Interestingly, the c.207_208dupTA mutation had no effect on the coding sequence, pre-mRNA splicing or the level of expression of the SMARCB1 isoform 2. Furthermore, SMARCB1 isoforms harboring a premature termination codon were largely eliminated via the nonsense-mediated mRNA decay pathway. Our results highlight the importance of RNA-based studies to characterize SMARCB1 germline mutations in order to determine their impact on protein expression and gain further insight into the genetic basis of conditions associated with SMARCB1 mutations.

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disease caused by mutations in the NF1 gene. The mutation rate of NF1 is one of the highest known for human genes and the mutational analysis has revealed a wide variety of changes, a significant proportion of which affect normal pre-mRNA splicing. Here, we describe two truncating mutations in exon 37 of NF1, the recurrent c.6792C>A and the novel c.6799C>T change, that occur in cis and segregate with NF1 in a large family. The double mutation induces defective splicing of exon 37 and thus, we performed quantitative comparisons of transcripts harboring single (c.6792C>G or c.6792C>A) and double (c.6792C>A and c.6799C>T) mutations to assess their effects on exon 37 splicing. Skipping of exon 37 was greater and there were fewer mutant full-length transcripts in samples with the double mutation than in those carrying single mutations. Thus, the combination of the c.6792C>A and c.6799C>T mutations augmented exon 37 skipping. These findings suggest that, in addition to the previously described exonic splicing enhancer in the c.6791_6795 region, c.6799 lies within an additional regulatory element that influences the splicing of exon 37.

Neurofibromatosis type 1 (NF1) is one of the most common human hereditary disorders, predisposing individuals to the development of benign and malignant tumors in the nervous system, as well as other clinical manifestations. NF1 is caused by heterozygous mutations in the NF1 gene and around 25% of the pathogenic changes affect pre-mRNA splicing. Since the molecular mechanisms affected by these mutations are poorly understood, we have analyzed the splicing mutations identified in exon 9 of NF1, which is particularly prone to such changes, to better define the possible splicing regulatory elements. Using a minigene approach, we studied the effect of five splicing mutations in this exon described in patients. These highlighted three regulatory motifs within the exon. An in vivo splicing analysis of an extensive collection of changes generated in the minigene demonstrated that the CG motif at c.910-911 is critical for the recognition of exon 9. We also found that the GC motif at c.945-946 is involved in exon recognition through SRSF2 and that this motif is part of a Composite Exon Splicing Regulatory Element made up of physically overlapping enhancer and silencer elements. Finally, through an in vivo splicing analysis and in vitro binding assays, we demonstrated that the c.1007G>A mutation creates an Exonic Splicing Silencer element that binds the hnRNPA1 protein. The complexity of the splicing regulatory elements present in exon 9 is most likely responsible for the fact that mutations in this region represent 25% of all exonic changes that affect splicing in the NF1 gene.