Hybrid Minigene Assay: An Efficient Tool to Characterize mRNA Splicing Profiles of <i>NF1</i> Variants
Neurofibromatosis type 1 (NF1) is caused by heterozygous loss of function mutations in the <i>NF1</i> gene. Although patients are diagnosed according to clinical criteria and few genotype-phenotype correlations are known, molecular analysis remains important. <i>NF1</i> displ...
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MDPI AG
2021-02-01
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| Series: | Cancers |
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| Online Access: | https://www.mdpi.com/2072-6694/13/5/999 |
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| author | Valeria Morbidoni Elisa Baschiera Monica Forzan Valentina Fumini Dario Seif Ali Gianpietro Giorgi Lisa Buson Maria Andrea Desbats Matteo Cassina Maurizio Clementi Leonardo Salviati Eva Trevisson |
| author_facet | Valeria Morbidoni Elisa Baschiera Monica Forzan Valentina Fumini Dario Seif Ali Gianpietro Giorgi Lisa Buson Maria Andrea Desbats Matteo Cassina Maurizio Clementi Leonardo Salviati Eva Trevisson |
| author_sort | Valeria Morbidoni |
| collection | DOAJ |
| description | Neurofibromatosis type 1 (NF1) is caused by heterozygous loss of function mutations in the <i>NF1</i> gene. Although patients are diagnosed according to clinical criteria and few genotype-phenotype correlations are known, molecular analysis remains important. <i>NF1</i> 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 <i>NF1</i> variants on splicing. We investigated 29 intronic and exonic <i>NF1</i> 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 <i>de novo</i> 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 <i>NF1</i> variants potentially affecting splicing and to detect hypomorphic effects that might have phenotypic consequences, avoiding the requirement of patient’s RNA. |
| first_indexed | 2024-03-09T06:15:25Z |
| format | Article |
| id | doaj.art-34f83f27538e4374b1a5bc2b2e1eab13 |
| institution | Directory Open Access Journal |
| issn | 2072-6694 |
| language | English |
| last_indexed | 2024-03-09T06:15:25Z |
| publishDate | 2021-02-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Cancers |
| spelling | doaj.art-34f83f27538e4374b1a5bc2b2e1eab132023-12-03T11:53:42ZengMDPI AGCancers2072-66942021-02-0113599910.3390/cancers13050999Hybrid Minigene Assay: An Efficient Tool to Characterize mRNA Splicing Profiles of <i>NF1</i> VariantsValeria Morbidoni0Elisa Baschiera1Monica Forzan2Valentina Fumini3Dario Seif Ali4Gianpietro Giorgi5Lisa Buson6Maria Andrea Desbats7Matteo Cassina8Maurizio Clementi9Leonardo Salviati10Eva Trevisson11Clinical Genetics Unit, Department of Women’s and Children’s Health, University of Padova, 35128 Padova, ItalyClinical Genetics Unit, Department of Women’s and Children’s Health, University of Padova, 35128 Padova, ItalyClinical Genetics Unit, Department of Women’s and Children’s Health, University of Padova, 35128 Padova, ItalyClinical Genetics Unit, Department of Women’s and Children’s Health, University of Padova, 35128 Padova, ItalyClinical Genetics Unit, Department of Women’s and Children’s Health, University of Padova, 35128 Padova, ItalyClinical Genetics Unit, Department of Women’s and Children’s Health, University of Padova, 35128 Padova, ItalyClinical Genetics Unit, Department of Women’s and Children’s Health, University of Padova, 35128 Padova, ItalyClinical Genetics Unit, Department of Women’s and Children’s Health, University of Padova, 35128 Padova, ItalyClinical Genetics Unit, Department of Women’s and Children’s Health, University of Padova, 35128 Padova, ItalyClinical Genetics Unit, Department of Women’s and Children’s Health, University of Padova, 35128 Padova, ItalyClinical Genetics Unit, Department of Women’s and Children’s Health, University of Padova, 35128 Padova, ItalyClinical Genetics Unit, Department of Women’s and Children’s Health, University of Padova, 35128 Padova, ItalyNeurofibromatosis type 1 (NF1) is caused by heterozygous loss of function mutations in the <i>NF1</i> gene. Although patients are diagnosed according to clinical criteria and few genotype-phenotype correlations are known, molecular analysis remains important. <i>NF1</i> 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 <i>NF1</i> variants on splicing. We investigated 29 intronic and exonic <i>NF1</i> 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 <i>de novo</i> 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 <i>NF1</i> variants potentially affecting splicing and to detect hypomorphic effects that might have phenotypic consequences, avoiding the requirement of patient’s RNA.https://www.mdpi.com/2072-6694/13/5/999neurofibromatosis type 1<i>NF1</i>splicingminigenehypomorphicleaky splicing |
| spellingShingle | Valeria Morbidoni Elisa Baschiera Monica Forzan Valentina Fumini Dario Seif Ali Gianpietro Giorgi Lisa Buson Maria Andrea Desbats Matteo Cassina Maurizio Clementi Leonardo Salviati Eva Trevisson Hybrid Minigene Assay: An Efficient Tool to Characterize mRNA Splicing Profiles of <i>NF1</i> Variants Cancers neurofibromatosis type 1 <i>NF1</i> splicing minigene hypomorphic leaky splicing |
| title | Hybrid Minigene Assay: An Efficient Tool to Characterize mRNA Splicing Profiles of <i>NF1</i> Variants |
| title_full | Hybrid Minigene Assay: An Efficient Tool to Characterize mRNA Splicing Profiles of <i>NF1</i> Variants |
| title_fullStr | Hybrid Minigene Assay: An Efficient Tool to Characterize mRNA Splicing Profiles of <i>NF1</i> Variants |
| title_full_unstemmed | Hybrid Minigene Assay: An Efficient Tool to Characterize mRNA Splicing Profiles of <i>NF1</i> Variants |
| title_short | Hybrid Minigene Assay: An Efficient Tool to Characterize mRNA Splicing Profiles of <i>NF1</i> Variants |
| title_sort | hybrid minigene assay an efficient tool to characterize mrna splicing profiles of i nf1 i variants |
| topic | neurofibromatosis type 1 <i>NF1</i> splicing minigene hypomorphic leaky splicing |
| url | https://www.mdpi.com/2072-6694/13/5/999 |
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