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. 2018 Oct 5;25(1):72.
doi: 10.1186/s12929-018-0474-9.

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

Yah-Huei Wu-Chou  1 , Tzu-Chao Hung  2 , Yin-Ting Lin  2 , Hsing-Wen Cheng  2 , Ju-Li Lin  3 , Chih-Hung Lin  4 , Chung-Chih Yu  4 , Kuo-Ting Chen  4 , Tu-Hsueh Yeh  5 , Yu-Ray Chen  6
Affiliations

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

Yah-Huei Wu-Chou et al. J Biomed Sci. .

Abstract

Background: Neurofibromatosis type 1 (NF1) is a dominantly inherited tumor predisposition syndrome that targets the peripheral nervous system. It is caused by mutations of the NF1 gene which serve as a negative regulator of the cellular Ras/MAPK (mitogen-activated protein kinases) signaling pathway. Owing to the complexity in some parts of clinical diagnoses and the need for better understanding of its molecular relationships, a genetic characterization of this disorder will be helpful in the clinical setting.

Methods: In this study, we present a customized targeted gene panel of NF1/KRAS/BRAF/p53 and SPRED1 genes combined with Multiple Ligation-Dependent Probe Amplification analysis for the NF1 mutation screening in a cohort of patients clinically suspected as NF1.

Results: In this study, we identified 73 NF1 mutations and two BRAF novel variants from 100 NF1 patients who were suspected as having NF1. These genetic alterations are heterogeneous and distribute in a complicated way without clustering in either cysteine-serine-rich domain or within the GAP-related domain. We also detected fifteen multi-exon deletions within the NF1 gene by MLPA Analysis.

Conclusions: Our results suggested that a genetic screening using a NGS panel with high coverage of Ras-signaling components combined with Multiple Ligation-Dependent Probe Amplification analysis will enable differential diagnosis of patients with overlapping clinical features.

Keywords: Genetic counseling; MLPA; Neurofibromatosis type 1; RASopathies; Targeted NGS.

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Conflict of interest statement

Ethics approval and consent to participate

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards. Ethics approval was obtained by the institutional review board (102-0226A3) at the Chang Gung Memorial Hospital. Informed consent was individually obtained from all participants included in the study.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures

Fig. 1
Fig. 1
Details of the 73 NF1 genetic variations identified by NGS targeted gene sequencing. The position of genetic variations detected in the NF1 gene from each patient is shown and their relationship to a possible defect of NF1 gene was also included. Known functional domains of Neurofibromin: CSRD > cysteine–serine-rich domain; GRD > GTPase-activating protein-related domain; SEC14/PH > SEC14 domain and pleckstrin homology (PH) domain; CTD > Carboxy-terminal domain; SBD > Syndecan-binding domain
Fig. 2
Fig. 2
Some represented results of Sanger sequencing at the mutation site with blood sample
Fig. 3
Fig. 3
Examples of multi-exon deletions detected by multiplex ligation-dependent probe amplification. In this study, we used the Coffalyser program (version 3.5) for peak area normalization and gene dosage calculation. Two copies of the genome have a relative peak area value of approximately 1.0. A reduction in the peak area value to < 0.7 indicates the occurrence of a deletion
See this image and copyright information in PMC

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