Hybrid Minigene Assay: An Efficient Tool to Characterize mRNA Splicing Profiles of NF1 Variants

SIMPLE SUMMARY: Heterozygous inactivating mutations in NF1 cause neurofibromatosis type 1, an autosomal dominant neurocutaneous disorder that shows variable clinical expressivity. Mutational spectrum is wide with a proportion of variants acting on splicing. Molecular diagnosis mostly relies on a combination of NF1 sequencing with multiplex ligation-dependent probe amplification (MLPA) analysis, but validation may be difficult. We employed a minigene system to examine a series of NF1 variants identified in patients. Our system allowed to assess the effect(s) on splicing for all the 29 variants we investigated, revealing multiple mechanisms of splicing alterations for some of them. One NF1 variant identified in a patient with NF1 allowed the production of residual levels of wild-type transcript. Our in vitro minigene system proved to be a fast and reliable method, which may be easily applied to validate NF1 variants, define their molecular mechanism(s), and detect hypomorphic effects of the variations. ABSTRACT: Neurofibromatosis type 1 (NF1) is caused by heterozygous loss of function mutations in the NF1 gene. Although patients are diagnosed according to clinical criteria and few genotype-phenotype correlations are known, molecular analysis remains important. NF1 displays allelic heterogeneity, with a high proportion of variants affecting splicing, including deep intronic alleles and changes outside the canonical splice sites, making validation problematic. Next Generation Sequencing (NGS) technologies integrated with multiplex ligation-dependent probe amplification (MLPA) have largely overcome RNA-based techniques but do not detect splicing defects. A rapid minigene-based system was set up to test the effects of NF1 variants on splicing. We investigated 29 intronic and exonic NF1 variants identified in patients during the diagnostic process. The minigene assay showed the coexistence of multiple mechanisms of splicing alterations for seven variants. A leaky effect on splicing was documented in one de novo substitution detected in a sporadic patient with a specific phenotype without neurofibromas. Our splicing assay proved to be a reliable and fast method to validate novel NF1 variants potentially affecting splicing and to detect hypomorphic effects that might have phenotypic consequences, avoiding the requirement of patient’s RNA.