Detection of novel NF1 mutations and rapid mutation prescreening with Pyrosequencing

Abstract

Neurofibromatosis type 1 (NF1) is caused by mutations in the neurofibromin (NF1) gene. Mutation analysis of NF1 is complicated by its large size, the lack of mutation hotspots, pseudogenes and frequent de novo mutations. Additionally, the search for NF1 mutations on the mRNA level is often hampered by nonsense-mediated mRNA decay (NMD) of the mutant allele. In this study we searched for mutations in a cohort of 38 patients and investigated the relationship between mutation type and allele-specific transcription from the wild-type versus mutant alleles. Quantification of relative mRNA transcript numbers was done by Pyrosequencing, a novel real-time sequencing method whose signals can be quantified very accurately. We identified 21 novel mutations comprising various mutation types. Pyrosequencing detected a definite relationship between allelic NF1 transcript imbalance due to NMD and mutation type in 24 of 29 patients who all carried frame-shift or nonsense mutations. NMD was absent in 5 patients with missense and silent mutations, as well as in 4 patients with splice-site mutations that did not disrupt the reading frame. Pyrosequencing was capable of detecting NMD even when the effects were only moderate. Diagnostic laboratories could thus exploit this effect for rapid prescreening for NF1 mutations as more than 60% of the mutations in this gene disrupt the reading frame and are prone to NMD.

Allele-Specific Expression of APC in Adenomatous Polyposis Families

“…As has been reported for MSH2 , MLH1 , BRCA1-2 and NF1 7-11, the nuclear scanning NMD model loosely fits with our observations for the APC gene. One of the exceptions is a mutation at intron 14 that creates a skipping of exon 14 and a PTC at the very beginning of exon 15 (data not shown) that associates with imbalanced ASE.…”

“…In other cancer predisposition genes such as MSH2 , MLH1 , BRCA1 , BRCA2 and NF1 , studies at the RNA level have shown that mutations causing a premature termination codon (PTC) usually trigger nonsense-mediated decay (NMD) of the mRNA7-11. This mRNA surveillance mechanism reduces the abundance of premature stop-codon-harboring mRNA and of the corresponding truncated proteins.…”

Development of a practical NF1 genetic testing method through the pilot analysis of five Japanese families with neurofibromatosis type 1

“…Additionally, 10 or more NF1 pseudogenes are present in the human genome, and these highly homologous DNA sequences prevent accurate NF1 mutation analysis. Furthermore, at the RNA level, mutation analysis is often hampered by nonsense-mediated mRNA decay (NMD) of the mutant allele [10]. Problems with NMD can be overcome using the protein truncation test (PTT) [11], but the PTT is technically complicated and lacks sufficient sensitivity.…”

A Novel Deep Intronic Mutation Introducing a Cryptic Exon Causing Neurofibromatosis Type 1 in a Family with Highly Variable Phenotypes: A Case Study

“…Literature search revealed 19 different deep intronic splice mutations reported so far, totally 20 when including the one found in the present study. The results with references are shown in Table 1 [4,16,20,[22][23][24][25][26][27][28][29][30][31][32].…”