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Pathogenic neurofibromatosis type 1 (NF1) RNA splicing resolved by targeted RNAseq

ABSTRACT: Neurofibromatosis type 1 (NF1) is caused by loss-of-function variants in the NF1 gene. Approximately 10% of these variants affect RNA splicing and are either missed by conventional DNA diagnostics or are misinterpreted by in silico splicing predictions. Therefore, a targeted RNAseq-based approach was designed to detect pathogenic RNA splicing and associated pathogenic DNA variants. For this method RNA was extracted from lymphocytes, followed by targeted RNAseq. Next, an in-house developed tool (QURNAs) was used to calculate the enrichment score (ERS) for each splicing event. This method was thoroughly tested using two different patient cohorts with known pathogenic splice-variants in NF1. In both cohorts all 56 normal reference transcript exon splice junctions, 24 previously described and 45 novel non-reference splicing events were detected. Additionally, all expected pathogenic splice-variants were detected. Eleven patients with NF1 symptoms were subsequently tested, three of which have a known NF1 DNA variant with a putative effect on RNA splicing. This effect could be confirmed for all 3. The other eight patients were previously without any molecular confirmation of their NF1-diagnosis. A deep-intronic pathogenic splice variant could now be identified for two of them (25%). These results suggest that targeted RNAseq can be successfully used to detect pathogenic RNA splicing variants in NF1.

SUBMITTER: Koster R

PROVIDER: S-EPMC8593033 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Pathogenic Neurofibromatosis type 1 (NF1) RNA splicing resolved by targeted RNAseq

Project description:Neurofibromatosis type 1 (NF1) is caused by loss-of-function variants in the NF1 gene. Approximately 10% of these variants affect RNA splicing and are either missed by conventional DNA diagnostics or are misinterpreted by in silico splicing predictions. Therefore, a targeted RNAseq-based approach was designed to detect pathogenic RNA splicing and associated pathogenic DNA variants. an in-house developed tool (QURNAS) was used to calculate the enrichment score (ERS) for each splicing event. RNA enrichment of NF1 and SPRED1 was done using SPET (NUGEN - NF1 only) and using SureSelect (Agilent - NF1 and SPRED1).

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NF1 Gene Novel Splicing Mutations in a Chinese Family with Neurofibromatosis Type 1: Case Series.

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Five novel NF1 gene pathogenic variants in 10 different Chinese families with neurofibromatosis type 1.

Project description:BackgroundNeurofibromatosis type 1 (NF1) is an autosomal dominant disorder with equal sex incidence that is characterized by neurofibromas, café-au-lait macules, axillary freckling, optic pathway tumor, distinctive osseous lesion, and iris Lisch nodules. Inactivating variants in the NF1 gene have been identified to be correlated with NF1. This tumor suppressor gene is located at 17q11.2.MethodsTen affected NF1 probands and their available relatives from 10 unrelated Chinese families with neurofibromatosis type 1 were clinically studied. All of these probands mainly complained of osseous lesions. PCR was used to analyze and sequence the variants. We collected both laboratory and radiological information.ResultsWe detected five novel pathogenic variants including two de novo variants in these 10 families: one missense variant, p.Cys709Arg(c.2125T>C), in exon 18 and four frameshift variants: p.Leu1459Profs*2(c.4436dupT) in exon 34; p.Lys99Argfs*4(c.296delA) in exon 4; p.Leu762Cysfs*2(c.2283delA) in exon 19; and p.Leu1522Ilefs*53(c.4562_4563dupAT) in exon 34.ConclusionNovel pathogenic variants in the NF1 gene in these families correlated with the phenotype and genotype and explained the clinical manifestations of these patients. The results help us to understand the genetic basis of patients with neurofibromatosis type 1 in China. Our study expands the pathogenic variant spectrum of the NF1 gene and may be helpful in genetic counseling and prenatal genetic diagnosis.

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Somatic neurofibromatosis type 1 (NF1) inactivation characterizes NF1-associated pilocytic astrocytoma.

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Targeted exon skipping of <i>NF1</i> exon 17 as a therapeutic for neurofibromatosis type I.

Project description:We investigated the feasibility of utilizing an exon-skipping approach as a genotype-dependent therapeutic for neurofibromatosis type 1 (NF1) by determining which NF1 exons might be skipped while maintaining neurofibromin protein expression and GTPase activating protein (GAP)-related domain (GRD) function. Initial in silico analysis predicted exons that can be skipped with minimal loss of neurofibromin function, which was confirmed by in vitro assessments utilizing an Nf1 cDNA-based functional screening system. Skipping of exons 17 or 52 fit our criteria, as minimal effects on protein expression and GRD activity were noted. Antisense phosphorodiamidate morpholino oligomers (PMOs) were utilized to skip exon 17 in human cell lines with patient-specific pathogenic variants in exon 17, c.1885G>A, and c.1929delG. PMOs restored functional neurofibromin expression. To determine the in vivo significance of exon 17 skipping, we generated a homozygous deletion of exon 17 in a novel mouse model. Mice were viable and exhibited a normal lifespan. Initial studies did not reveal the presence of tumor development; however, altered nesting behavior and systemic lymphoid hyperplasia was noted in peripheral lymphoid organs. Alterations in T and B cell frequencies in the thymus and spleen were identified. Hence, exon skipping should be further investigated as a therapeutic approach for NF1 patients with pathogenic variants in exon 17, as homozygous deletion of exon 17 is consistent with at least partial function of neurofibromin.

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A novel neurofibromatosis type 1 (NF1) mutation in a patient with NF1 and pheochromocytoma.

Project description: Not available

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Two Novel NF1 Pathogenic Variants Causing the Creation of a New Splice Site in Patients With Neurofibromatosis Type I.

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