Long RT-PCR of the entire 8.5-kb NF1 open reading frame and mutation detection on agarose gels.

Abstract

Previous approaches to mutation detection in mRNA from the neurofibromatosis 1 (NF1) locus have required the PCR amplification of five or more overlapping cDNA segments to screen the entire 8.5-kb open reading frame (ORF). Systematically, these assays do not detect deletions that span the region of overlap (usually 1-3 exons) of any two consecutive target segments. In such cases, amplification from the mutant region of the disease-causing allele fails because binding sites for the PCR primers are missing, but amplification from the normal allele proceeds, yielding only the normal product. To alleviate this problem, we have developed a protocol to reverse transcribe and amplify the entire protein-coding sequence of NF1 as a single PCR product, starting with total RNA from lymphoblast cell lines or from whole blood. The 8.7-kb RT-PCR product was prepared from nine NF1 patients with known deletions or insertions, ranging in size from a 30-bp deletion within 1 exon to a 2.4-kb deletion that removes 12 exons. Agarose gel analysis of the initial products detected deletions as small as 341 bp. Restriction endonuclease digestion with Asel and Fspl, followed by agarose gel electrophoresis, revealed the predicted abnormal bands in all nine patients. All mutant bands were identified readily by observers with no knowledge of the patients' mutations. This simple assay should detect a great variety of insertion/deletion mutations in the NF1mRNA internal to the primer binding sites, including all possible single and multiple exon dropouts and approximately 30% of all previously reported NF1 mutations.

High‐yield identification of pathogenic NF1 variants by skin fibroblast transcriptome screening after apparently normal diagnostic DNA testing

“…An established effective approach for NF1 variant detection is cDNA‐based screening of lymphocyte RNA (Ars et al, 2000 ; Martinez et al, 1996 ; Messiaen et al, 2000 ; Serra et al, 2001 ; Wimmer et al, 2011 , 2020 ; Zatkova et al, 2004 ). An advantage of this RNA‐first approach is that only one blood sample is required.…”

“…These individuals are often referred to as NF1 “no mutation identified” (NMI). In some laboratories, particularly those with a focus on NF1, routine DNA screening by polymerase chain reaction (PCR)/Sanger sequencing and multiplex ligation probe amplification (MLPA) has been complemented or replaced entirely by RNA‐ and next‐generation sequencing (NGS)‐ based techniques that increase the diagnostic yield to >95% by identifying variants that affect pre‐mRNA splicing and/or are present at a low frequency due to mosaicism (Ars et al, 2000 ; Evans et al, 2016 ; Koster et al, 2021 ; Martinez et al, 1996 ; Messiaen et al, 2000 ; Serra et al, 2001 ; Wimmer et al, 2011 , 2020 ; Zatkova et al, 2004 ). Unfortunately, such a specialized, focused approach is not available in all diagnostic laboratories.…”

Molecular and Functional Properties of the Human α1G Subunit That Forms T-type Calcium Channels

“…Long Range RT-PCR for Native Isoform Identification-A search for native isoforms of the ␣ 1G subunit was further performed using long range RT-PCR (23) and sequence examination of the cDNA fragment covering from nt 2840 (domain II) to nt 6272 (C-terminal region), according to AF126966. The size of this fragment varied from ϳ3.4 to ϳ3.8 kb, as a function of the ␣ 1G isoform detected.…”

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

“…In contrast to laboratories that specialize in NF1 variant detection and classification using patient RNA [21,39], our diagnostic laboratory performs molecular screening primarily on DNA samples because direct analysis of RNA was not considered practical for routine screening in our setting [18]. The in vitro exon trap experiments provided a useful screen for identifying NF1 variants likely to affect splicing, without having to resample patients.…”