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

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.

RAD51D Aberrant Splicing in Breast Cancer: Identification of Splicing Regulatory Elements and Minigene-Based Evaluation of 53 DNA Variants

“…Despite the fact that assays using patient-derived RNA are considered the most suitable strategy to evaluate splicing outcomes, minigene assays have proved to be a useful, simple and robust approach to assess potential spliceogenic variants [ 12 , 40 ]. This method presents several significant advantages such as (1) analysis of single allele events without the meddling of the WT allele; (2) accurate quantification of isoforms by inhibiting the nonsense-mediated decay; (3) high versatility, allowing the study of different variants with a single minigene; (4) high reproducibility of physiological and pathological splicing patterns; (5) analysis in a cell type relevant for the disease.…”

“…Unfortunately, the lack of accurate in silico predictors makes functional assays vital to doing so. Next, splicing functional assays, either from patient or minigene RNAs, provide critical data to evaluate the pathogenicity of a particular genetic variant [ 11 , 12 , 13 , 14 , 15 ].…”

Natural history and genetic study of LAMA2-related muscular dystrophy in a large Chinese cohort

“…Moreover, splicing variant tended to be associated with a relatively mild phenotype in LAMA2 -CMD. Leaky splicing was reported to reduce the symptoms and self-improve clinical phenotype in some genetic diseases such as spinal muscular atrophy and neurofibromatosis type 1 [ 41 , 42 ]. The impact of the different splicing variants might be related with a leaky splicing or other mechanisms which should be further studied.…”

Splicing Outcomes of 5′ Splice Site GT>GC Variants That Generate Wild-Type Transcripts Differ Significantly Between Full-Length and Minigene Splicing Assays

“…RNA analysis using either patient blood cells or immortalized lymphoblastoid cells represents an alternative option, providing that the gene of interest is normally expressed in these cells (Wai et al, 2020). In case of the non-feasibility of both approaches, a cell culture-based minigene splicing assay has often been devised (for some most recent examples, see Damasio et al, 2021;Hao et al, 2021;Kim et al, 2021;Kortum et al, 2021;Le Tertre et al, 2021;Morbidoni et al, 2021;Qian et al, 2021;Saint-Martin et al, 2021;Torrado et al, 2021).…”