Functional Assays Combined with Pre-mRNA-Splicing Analysis Improve Variant Classification and Diagnostics for Individuals with Neurofibromatosis Type 1 and Legius Syndrome

Neurofibromatosis type 1 (NF1) and Legius syndrome (LS) are caused by inactivating variants in NF1 and SPRED1. NF1 encodes neurofibromin (NF), a GTPase activating protein (GAP) for RAS, that interacts with the SPRED1 product, Sprouty-related protein with an EVH (Ena/Vasp homology) domain 1 (SPRED1). Establishing a clinical and molecular diagnosis of NF1 or LS can be challenging due to the phenotypic diversity, the size and complexity of the NF1 and SPRED1 loci and uncertainty over the effects of variants on pre-mRNA splicing and NF/SPRED1 function. The purpose of this work was to improve NF1 and SPRED1 variant classification. To help establish the pathogenicity of NF1 and SPRED1 variants identified in individuals with NF1 or LS, we employed 4 assays: (i) analysis of patient RNA by RT-PCR; (ii) in vitro exon trap analysis of NF1 pre-mRNA splicing; (iii) in vitro analysis of NF RAS GAP activity; and (iv) in vitro analysis of the NF-SPRED1 interaction. In 69/105 (66%) cases we obtained evidence to support variant pathogenicity according to American College of Medical Genetics guidelines, demonstrating the utility of functional approaches for NF1 and SPRED1 variant classification and NF and LS diagnostics.

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“…A preprint of this manuscript has previously been published [46] (https://yvm2020.authorea.com/doi/full/10 .22541/au.165217208.81458305/v1).…”

Section: Data Availabilitymentioning

confidence: 99%

Functional assays combined with pre-mRNA splicing analysis improve variant classification and diagnostics for individuals with Neurofibromatosis type 1 and Legius syndrome.

Nellist

Douben

Hoogeveen‐Westerveld

et al. 2022

Preprint

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Genotype–Phenotype Correlation in Neurofibromatosis Type 1: Evidence for a Mild Phenotype Associated with Splicing Variants Leading to In-Frame Skipping of NF1 Exon 24 [19a]

Chen,

Fu,

Koczkowska

et al. 2024

Cancers

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Neurofibromatosis type 1 (NF1) is an autosomal dominant neurocutaneous disorder caused by loss-of-function variants in the NF1 gene. As of 20 November 2023, over 5000 distinct pathogenic or likely pathogenic variants have been reported in public databases. However, only a few NF1 genotype–phenotype correlations have been established so far. In this study, we present findings on 40 individuals with NF1, comprising 26 unrelated probands and 14 affected relatives, who carry one of nine NF1 heterozygous pathogenic splicing variants, all of which result in the in-frame skipping of exon 24 [19a] (NM_000267.3:r.3114_3197del, p.Asn1039_Arg1066del). These variants include c.3114-2A>G, c.3114-1G>A, c.3196A>G, c.3197G>A, c.3197G>T, c.3197+1G>A, c.3197+1G>T, c.3197+2T>C, and c.3197+3A>T. Among individuals with these variants, none exhibit externally visible plexiform neurofibromas, histopathologically confirmed cutaneous or subcutaneous neurofibromas, symptomatic spinal neurofibromas, or symptomatic optic pathway gliomas. The most prevalent, and sometimes sole, clinical feature observed in this cohort is multiple café-au-lait macules, with or without skinfold freckles: 85% and 60.5% of the individuals display six or more café-au-lait macules and freckles, respectively. In comparison to established NF1 genotype–phenotype correlations, these patients demonstrate highly similar clinical presentations to those associated with the NF1 pathogenic variant c.2970_2972del (p.Met992del), known for resulting in the mildest clinical features. Despite the generally mild phenotype, cognitive impairment, developmental delay, and/or learning difficulties are still observed in 33.3% of these patients, suggesting that learning challenges remain a prominent aspect of the phenotypic presentation in these individuals and necessitate specialized care. This newly established genotype–phenotype correlation will assist clinicians in improving the management of patients harboring NF1 exon 24 [19a] skipping variants and provide a new therapeutic target for NF1 treatment.

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Biomarker Landscape in RASopathies

Ferrito,

Báez-Flores,

Rodríguez-Martín

et al. 2024

IJMS

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RASopathies are a group of related genetic disorders caused by mutations in genes within the RAS/MAPK signaling pathway. This pathway is crucial for cell division, growth, and differentiation, and its disruption can lead to a variety of developmental and health issues. RASopathies present diverse clinical features and pose significant diagnostic and therapeutic challenges. Studying the landscape of biomarkers in RASopathies has the potential to improve both clinical practices and the understanding of these disorders. This review provides an overview of recent discoveries in RASopathy molecular profiling, which extend beyond traditional gene mutation analysis. mRNAs, non-coding RNAs, protein expression patterns, and post-translational modifications characteristic of RASopathy patients within pivotal signaling pathways such as the RAS/MAPK, PI3K/AKT/mTOR, and Rho/ROCK/LIMK2/cofilin pathways are summarized. Additionally, the field of metabolomics holds potential for uncovering metabolic signatures associated with specific RASopathies, which are crucial for developing precision medicine. Beyond molecular markers, we also examine the role of histological characteristics and non-invasive physiological assessments in identifying potential biomarkers, as they provide evidence of the disease’s effects on various systems. Here, we synthesize key findings and illuminate promising avenues for future research in RASopathy biomarker discovery, underscoring rigorous validation and clinical translation.

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Functional Assays Combined with Pre-mRNA-Splicing Analysis Improve Variant Classification and Diagnostics for Individuals with Neurofibromatosis Type 1 and Legius Syndrome

Cited by 3 publications

References 48 publications

Functional assays combined with pre-mRNA splicing analysis improve variant classification and diagnostics for individuals with Neurofibromatosis type 1 and Legius syndrome.

Functional assays combined with pre-mRNA splicing analysis improve variant classification and diagnostics for individuals with Neurofibromatosis type 1 and Legius syndrome.

Genotype–Phenotype Correlation in Neurofibromatosis Type 1: Evidence for a Mild Phenotype Associated with Splicing Variants Leading to In-Frame Skipping of NF1 Exon 24 [19a]

Biomarker Landscape in RASopathies

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