Neurofibromatosis Type 1 Gene Mutation Analysis Using Sequence Capture and High-throughput Sequencing

Abstract

Neurofibromatosis type 1 syndrome (NF1) is caused by mutations in the NF1 gene. Availability of new sequencing technology prompted us to search for an alternative method for NF1 mutation analysis. Genomic DNA was isolated from saliva avoiding invasive sampling. The NF1 exons with an additional 50bp of flanking intronic sequences were captured and enriched using the SeqCap EZ Choice Library protocol. The captured DNA was sequenced with the Roche/454 GS Junior system. The mean coverages of the targeted regions were 41x and 74x in 2 separate sets of samples. An NF1 mutation was discovered in 10 out of 16 separate patient samples. Our study provides proof of principle that the sequence capture methodology combined with high-throughput sequencing is applicable to NF1 mutation analysis. Deep intronic mutations may however remain undetectable, and change at the DNA level may not predict the outcome at the mRNA or protein levels.

Distribution and Within-Family Specificity of Quantitative Autistic Traits in Patients with Neurofibromatosis Type I

“…To date, published reports of molecular genetic testing have identified more than 1400 pathogenic mutations of various types dispersed throughout the gene. 7 This degree of genetic diversity parallels its extraordinary variability in phenotypic expression and severity. Other than case reports, 8,9 there are no known associations between specific NF1 genotypes and autistic syndromes, and there are no published studies in which both genotype and autistic impairment in NF1 have been characterized simultaneously.…”

Hybridization Capture-Based Next-Generation Sequencing to Evaluate Coding Sequence and Deep Intronic Mutations in the NF1 Gene

“…Although some previous studies had evaluated the use of NGS technology to investigate mutations in the NF1 gene [3,7,16,17,18,19,20,21], only exons and conserved splice sites were sequenced, which is the approach commonly used by other investigators based on Sanger methodology.…”

“…As it occurs in other genes, the first exon of the NF1 gene presents higher GC content (71%), compared to the other exons (42%) [7]. It is known that coverage may be reduced in regions with GC contents outside of an optimum range of 40%–60% [25,26], and low coverage in exon 1 of the NF1 gene had been previously reported in other studies with NGS, using both PCR-based enrichment and hybridization-based targeted selection of the NF1 gene for library preparation [7,19]. Regarding the use of PCR-based enrichment, high/low GC content reduces the efficiency of PCR amplification.…”

“…Identification and characterization of NF1 gene mutations represent a considerable research and diagnostic challenge because of its large size, existence of a number of highly homologous pseudogenes located throughout the human genome (in chromosomes 2, 12, 14, 15, 18, 21, and 22), absence of mutational hotspots as well as the diversity of mutation types, including deep intronic splicing mutations [6,7]. The NF1 gene mutation detection rate in NF1 patients who meet the clinical diagnostic criteria is reported to be 50%–95% (reviewed by Upadhyaya [8]).…”

Clinical Characteristics of the Halo Phenomenon in Infants with Neurofibromatosis 1: A Case Series

“…Although 95% of patients with neurofibromatosis 1 (NF1) can be diagnosed by the age of 8 years using National Institutes of Health criteria (1), diagnosis in infants without a family history is sometimes difficult. Mutation analysis has recently been reported to be useful for early diagnosis, but it is often difficult to perform genetic diagnostics in daily medical practice (2). The presence of multiple café au lait macules (CALMs) on the entire body including the back is the most common symptom of NF1 in infants.…”