NF1 single and multi-exons copy number variations in neurofibromatosis type 1

Abstract

Neurofibromatosis type 1 (NF1) is caused by dominant loss-of-function mutations of the tumor suppressor NF1 containing 57 constitutive coding exons. A huge number of different pathogenic NF1 alterations has been reported. The aim of the present study was to evaluate the usefulness of a multiplex ligation-dependent probe amplification (MLPA) approach in NF1 patients to detect single and multi-exon NF1 gene copy number variations. A genotype-phenotype correlation was then performed in NF1 patients carrying these types of genetic alterations. Among 565 NF1 index cases from the French NF1 cohort, single and multi-exon deletions/duplications screening identified NF1 partial deletions/duplications in 22 patients (~4%) using MLPA analysis. Eight single exon deletions, 11 multiple exons deletions, 1 complex rearrangement and 2 duplications were identified. All results were confirmed using a custom array-CGH. MLPA and custom array-CGH allowed the identification of rearrangements that were missed by cDNA/DNA sequencing or microsatellite analysis. We then performed a targeted next-generation sequencing of NF1 that allowed confirmation of all 22 rearrangements. No clear genotype-phenotype correlations were found for the most clinically significant disease features of NF1 in patients with single and multi-exons NF1 gene copy number changes.

Genetic diagnosis of neurofibromatosis type 1: targeted next- generation sequencing with Multiple Ligation-Dependent Probe Amplification analysis

“…For patients in whom mutations were not detected by our NGS panel screening, we detected fifteen multi-exon deletions within the NF1 gene by Multiplex Ligation-Dependent Probe Amplification (MLPA) analysis (~ 15% of detected NF1 alterations). A multi-step mutation detection protocol has been used for over 95% of pathogenic NF1 mutations in different laboratories [ 15 – 21 , 26 – 28 ]. The NF1 mutations were detected in our study was in 92.6% (88/95) of the subjects when five patients who did not completely met the clinical diagnostic criteria were excluded.…”

“…Therefore, molecular diagnosis in NF1 should be of great value to confirm the diagnosis, particularly in the early childhood. However, the procedures for molecular diagnosis of NF1 are usually expensive, time-consuming, and labor-intensive [ 15 – 21 , 26 – 28 ]. The development of next-generation sequencing (NGS) technologies which allows for rapid identification of disease-causing mutations and high-risk alleles has recently been introduced into NF1 diagnosis [ 29 – 34 ].…”

qEva-CRISPR: a method for quantitative evaluation of CRISPR/Cas-mediated genome editing in target and off-target sites

“…Sensitivity to large deletions is the main property of the ligation-dependent probes that are routinely utilized in MLPA assays for large mutation detection [e.g. ( 27 , 29 , 49 , 50 )]. Moreover, the short region-specific sequence (usually ∼40 nt) of the qEva-CRISPR probes utilized in our study made them relatively insensitive to the single-nucleotide substitutions (SNPs) that commonly occur in the human genome as well as in other genomes.…”

“…The standard MLPA setup was validated and successfully used in hundreds of research and clinical studies for the analysis of large mutations in disease-related genes [e.g. ( 27 , 29 , 49 , 50 )]. (vii) The qEva-CRISPR strategy can be easily adapted to the target or off-targets of interest.…”

Neurofibromatosis Type 1 Molecular Diagnosis: The RNA Point of View

“…Evans and colleagues also underlined that sequencing at the cDNA level can increase mutation detection sensitivity due to the presence of deep intronic mutations, or exons deletions ( Imbard et al, 2015 ). Their screen for NF1 gene lesions identified ~ 96% of pathological mutations in patients presenting with typical NF1, using a two-step approach including a cDNA Sanger sequencing and copy number variations (CNV) study.…”