Breakpoint characterization of a novel NF1 multiexonic deletion: a case showing expression of the mutated allele

Abstract

Neurofibromatosis type 1 (NF1) is a common genetic disease caused by haploinsufficiency of the NF1 tumor-suppressor gene. Different pathogenetic mechanisms have been identified, with the majority (95%) causing intragenic lesions. Single or multiexon NF1 copy number changes occur in about 2% of patients, but little is known about the molecular mechanisms behind these intragenic deletions. We report here on the molecular characterization of a novel NF1 multiexonic deletion. The application of a multidisciplinary approach including multiplex ligation-dependent probe amplification, allelic segregation analysis, and fluorescent in situ hybridization allowed us to map the breakpoints in IVS27b and IVS48. Furthermore, the breakpoint junction was characterized by sequencing. Using bioinformatic analysis, we identified some recombinogenic motifs in close proximity to the centromeric and telomeric breakpoints and predicted the presence of a mutated messenger ribonucleic acid, which was deleted between exons 28 and 48 and encodes a neurofibromin that lacks some domains essential for its function. Through reverse transcriptase-polymerase chain reaction, the expression of the mutated allele was verified, showing the junction between exons 27b and 49 and, as expected, was not subjected to nonsense-mediated decay. Multiexonic deletions represent 2% of NF1 mutations, and until now, the breakpoint has been identified in only a few cases. The fine characterization of multiexonic deletions broadens the mutational repertoire of the NF1 gene, allowing for the identification of different pathogenetic mechanisms causing NF1.

Decoding NF1 Intragenic Copy-Number Variations

“…We also included two subjects with previously published NF1 CNVs with known breakpoints (IRO-1 and UNIMI-1) to investigate the nature and origin of their rearrangement mechanisms. 38,39 In total, 87 subjects with NF1 intragenic CNVs were investigated in this study. Subjects were originally referred for NF1 diagnostic testing.…”

“…Furthermore, we included two additional published NF1 CNVs (from IRO-1 and UNIMI-1). 38,39 We analyzed all 87 junction fragments for microhomology, potential to adopt non-B DNA structures, repetitive elements, and effect on coding sequence. We further inferred whether the CNV was present in mosaic form (versus constitutional) by evaluation of the cDNA and MLPA results and rearrangement mechanisms ( Table 1, Tables S1 and S3, and Figure S1).…”

“…We have characterized the likely rearrangement mechanism of these 85 CNVs along with two additional previously published NF1 CNVs. 38,39 Unlike recurrent rearrangements, mediated by flanking LCRs, NF1 intragenic CNVs originate by different rearrangement mechanisms and have diverse sizes and breakpoint positions. Furthermore, in addition to the loop within a 197-bp palindrome located in intron 40, four Alu elements located in introns 1, 2, 3, and 50 were identified as intragenic-rearrangement hotspots within NF1.…”