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

  • Yah-Huei Wu-Chou
  • Tzu-Chao Hung
  • Yin-Ting Lin
  • Hsing-Wen Cheng
  • Ju-Li Lin
  • Chih-Hung Lin
  • Chung-Chih Yu
  • Kuo-Ting Chen
  • Tu-Hsueh Yeh
  • Yu-Ray Chen
Open Access
Research

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

Neurofibromatosis type 1 RASopathies Targeted NGS MLPA Genetic counseling 

Abbreviations

CSRD

Cysteine–serine-rich domain

CTD

Carboxy-terminal domain

GAP-related domain

GTPase activating protein-related domain

GRD

GTPase-activating protein-related domain

HGMD

Human Gene Mutation Database

IGV

Integrative Genome Viewer

indels variants

insertions/deletions variants

ISP

Ion Sphere Particles

LOVD

Leiden Open Variation Database

MAF

Minor Allele Frequency

MLPA

Multiple Ligation-Dependent Probe Amplification

MRI

Magnetic Resonance Image

NCFC syndromes

Neuro-Cardio-Facio-Cutaneous syndromes

NF1

Neurofibromatosis type 1

NGS

Next-Generation Sequencing

PCR

Polymerase Chain Reaction

PH

pleckstrin homology domain

PolyPhen2

Polymorphism Phenotyping v2

Ras/MAPK signaling pathway

Ras/Mitogen-Activated Protein Kinases signaling pathway

SBD

Syndecan-binding domain

SEC14/PH

SEC14 domain and Pleckstrin Homology domain

SIFT

Sorting Intolerant from Tolerant

SNVs

Single Nucleotide Variants

Background

Neurofibromatosis type 1 (MIM# 162200) is a very common genetic disorder affecting approximately 1 in 3000–4000 individuals worldwide with the penetrance of the mutant gene being close to 100% by 5 years of age [1, 2, 3, 4]. Clinically, it is presented with the occurrence of Café-au-lait macules, Lisch nodules, axillary freckling and multiple neurofibromas. Phenotypically, Neurofibromatosis type 1 (NF1) patients have a very heterogeneous condition. Discrete dermal neurofibromas occur in almost all adults with NF1, and the number usually increases with age. If whole-body magnetic resonance imaging (MRI) is used, plexiform neurofibromas are detectable in at least half of NF1 patients. Other complications include learning disabilities, mental retardation, optic gliomas, certain bone abnormalities, CNS tumors, and an increased risk for certain malignancies [5, 6].

NF1 is caused by mutations of the NF1 gene which maps to chromosome 17q11.2. Many evidences have suggested NF1 as a tumor suppressor gene as inactivation of both NF1 alleles would reduce the control of cell proliferation and lead to tumorigenesis [7, 8]. The function of NF1 gene product, neurofibromin, is to stimulate the GTPase activity of the RAS protein and serve as a negative regulator of the cellular Ras/MAPK (mitogen-activated protein kinases) signaling pathway [7, 9, 10, 11]. Up to date, more than 1000 pathogenic allelic variants have been identified in the NF1 gene [The Human Gene Mutation Database (HGMD, Institute of Medical Genetics, Cardiff, http://www.hgmd.org/; Leiden Open Variation Database, LOVD: www.lovd.nl/NF1]. Most NF1 mutations are single-base substitutions, insertions, or deletions. Other mutations are single- or multi-exon deletions or duplications and microdeletions encompassing NF1 and its neighboring genes [12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22].

NF1 is a member of RAS-related disorders, which usually show similar clinical features in cutaneous signs, cardiac defects, developmental disabilities and neurocognitive impairment [23, 24, 25]. 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, 16, 17, 18, 19, 20, 21, 26, 27, 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, 30, 31, 32, 33, 34]. Owing to the complexity with some aspects of clinical diagnoses and the need for a better understanding of its molecular relationships, an extended genetic characterization of this disorder will be helpful in a clinical setting.

Methods

Patients and sample preparations

One hundred NF1 patients suspected as having NF1 by a clinical evaluation were recruited for this study. From each patient, 10 ml of whole blood samples were collected in EDTA-anticoagulant tube through the Linko Medical Center of the Chang Gung Memorial Hospital. Fifteen patients had a known family history of NF1. Ethical approval for this study was obtained by the institutional review board (102-0226A3) at Chang Gung Memorial Hospital. All participants provided written informed consent. Genomic DNA of each patient was then prepared using the PUREGENE DNA purification kit from GENTRA using standard protein precipitation procedures. The quality of the DNA was estimated using the Nano-Drop spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).

Candidate gene-targeted sequencing

A panel of five NF1-related genes including NF1 (NM_000267, 17q11.2), SPRED1 (NM_152594, 15q14), KRAS (NM_004985, 12p12.1), BRAF (NM_004333, 7q34), and p53 (NM_000546, 17p13.1) was initially created designed to capture, amplify, and sequence specific regions (including exons and splice junctions) of the genome for human cancer screening. The total length was 32.3 kb encompassing 296 amplicons, and the coverage was 507×. Adapter sequences were clonally amplified by emulsion PCR on the high-density array of micro-machined wells. In this study, we took the advantage of this gene panel for the germline mutation analysis of NF1 using the Ion Personal Genome Machine® (PGM™) Sequencer (Life technology).

Sample library preparation

A total of 100 indexed rapid prepared Ion AmpliSeq DNA libraries, starting from 100 ng of gDNA per sample, were prepared according to the manufacturer’s instructions. Template preparation and emulsion PCR and Ion Sphere Particles (ISP) enrichment were performed according to the manufacturer’s instructions. Following the purification and size selection using AMPure beads (Beckman Coulter, Brea, CA, USA), the size distribution of the DNA fragments was analyzed on the Agilent Bioanalyzer using High-Sensitivity DNA chip (Agilent Technologies Inc., Santa Clara, CA) and the quality checking of ion sphere particles for the prepared library was performed using Qubit 2.0 Fluorometer (Life Technologies). Enriched ISPs were prepared for sequencing using the Ion PGM 200 Sequencing Kit v2.0 and were loaded on an Ion 316 chip v2 or Ion 318 chip v2.

Data analysis

We used IT platform-specific pipeline software Torrent Suite, version 4.2, with the plug-in “variant caller” program (Life Technologies) to perform reference genome alignment, base calling, and filtering of poor signal reads. The Integrative Genome Viewer (IGV) (http://software.broadinstitute.org/software/igv/) was used for visualizing the status of each read alignment. The selected variants were classified as deleterious mutation by mutation type if they were identified as nonsynonymous, frameshift, or stopgain at the exonic region. ACMG Standards and Guidelines for the interpretation of sequence variants were followed in this study [35]. In an appropriate reference population, the pathogenic variant should have a frequency of much less than 1%. We removed all the common variants (Minor Allele Frequency, MAF > 1%) reported in the following public databases: 1000 Genomes Project (http://www.1000genomes.org/), dbSNP database and ClinVar database (https://www.ncbi.nlm.nih.gov/snp/; https://www.ncbi.nlm.nih.gov/clinvar/). Variants with amino acid changes were further examined for whether the changes were potentially damaging alterations using Sorting Tolerant From Intolerant (SIFT) and Polymorphism Phenotyping v2 (PolyPhen2) softwares, which can predict the possible impact of an amino acid substitution on the structure and function of a protein. The nomenclature of novel variants followed the rules of the Human Genome Variation Society (http://www.hgvs.org/mutnomen/). The genetic variants in the Human Gene Mutation Database (HGMD, Institute of Medical Genetics, Cardiff, http://www.hgmd.org/) and Leiden Open Variation Database (LOVD: www.lovd.nl/NF1) were also considered as references.

PCR amplification and sanger sequencing verification

We performed Sanger validation for all putatively pathogenic SNVs and indels variants on each detected patient (and their family members, if available) by PCR amplification and sequenced with Applied Biosystems 3730 Genetic Analyzer. PCR amplification was performed under standard conditions with 30 PCR cycles and 55°–60 °C annealing. PCR products were sequenced using the Big Dye Terminator cycle sequencing kit (Life Technologies) according to the manufacturer’s cycling conditions and analyzed on an Applied Biosystems 3730xl Automated Sequencer Genetic Analyzer (Life Technologies). Sequence alignments and analysis were further performed using the Autoassembler computer program (Life Technologies).

Multiplex ligation-dependent probe amplification (MLPA) analysis

We used SALSA P081/P082 NF1 MLPA kit (MRC Holland, Amsterdam, The Netherlands) to confirm and identify single and multiple exon deletions/duplications according to the manufacturer’s protocol. Each samples containing 100 ng of genomic DNA was used for overnight hybridization with the probemixes. After ligation and amplification were performed with FAM-labeled primers, the PCR products were analyzed on a Genetic Analyzer 3730 capillary electrophoresis system and interpreted using Genotyper version 2.0 (Applied Biosystems, CA, USA). In this study, we used the Coffalyser program (version 3.5) for peak area normalization and gene dosage calculation.

Results

Genetic alterations identified from a targeted NGS gene panel screening

A total of 100 individuals from 95 families who were clinically suspected as carrying NF1 were referred for this genetic testing. A brief summary of the clinical data collected for each patient is given in Table 1. Fifteen patients (15%) had a family history of NF1 in this cohort. Café-au-lait spots and Lisch nodules in the iris were observed in 93 and 19 patients, respectively. Cutaneous neurofibromas, plexiform neurofibromas and malignant peripheral nerve sheath tumors were identified in 32, 13, and 2, patients, respectively. Five patients had optic gliomas and two patients had brain tumors. Among these individuals, we have identified seventy-three NF1 mutations (Table 2) and two BRAF novel variants from a targeted NGS gene panel of NF1/KRAS/BRAF/p53 and SPRED1 analyses. SPRED1 genetic mutations were not detected in this study. Variants with amino acid changes were further examined to check if the changes were potentially damaging alterations using Sorting Intolerant from Tolerant (SIFT) algorithm and Polymorphism Phenotyping v2 (PolyPhen2) software, which can predict the possible impact of an amino acid substitution on the structure and function of a protein.
Table 1

Clinical features of 100 Taiwanese NF1 patients

Clinical features

Patientsa (%)

Café-au-lait spots

93 (93%)

Lisch nodules in the Iris

19 (19%)

Cutaneous neurofibroma

32 (32%)

Plexiform neurofibroma

13 (13%)

Malignant peripheral nerve sheath tumor

2 (2%)

Optic glioma

5 (5%)

Brain tumor

2 (2%)

Scoliosis

10 (10%)

Heart defects

8 (8%)

Learning disability

4 (4%)

Craniofacial disability

9 (9%)

Family history

15 (15%)

a11 patients are under 12 years old; male: female = 53:47

Table 2

NF1 Mutational profile of the 100 NF1 blood samples tested in NGS study

Patient

Codinga

Amino Acid Change

Variant Effect

NM_000267.3

SIFT

Polyphen2

Wu p001

c.492_495 del AACT/Het

p.Val166fs

Frameshift Deletion

Exon 5

  

Wu p002

c.5844C > A

p.Tyr1948Ter

nonsense

Exon 40

  

Wu p003

c.1466A > G, c.1400C > T, c.1448A > G, c.1513A > G

p.Tyr489Cys

missense

Exon 13

Tolerated

Benign

Wu p004

c.6855C > A

p.Tyr2285Ter

nonsense

Exon 46

Tolerated

 

Wu p006

c.2982_2982delT

p.Leu995fs

Frameshift Deletion

Exon 22

  

Wu p007

c.1105C > T

p.Gln369Ter

nonsense

Exon 10

Tolerated

 

Wu p009

c.7862_7862delC

p.Thr2621fs

Frameshift Deletion

Exon 54

  

Wu p010

c.5902C > T

p.Arg1968Ter

nonsense

Exon 40

Tolerated

 

Wup011

c.7152_7157del TAACTT

p.2384_2386del

Deletion

Exon 49

  

Wu p014

c.3113 + 1 G > A

.

splicing

Exon 23

  

Wu p015

c.4700C > G

p.Ser1567Ter

nonsense

Exon 36

Tolerated

 

Wu p017

c.487G > T

p.Glu163Ter

nonsense

Exon 5

  

Wu p018

c.6970C > T

p.Gln2324Ter

nonsense

Exon 47

Damaging

 
 

c.8386A > C

p.Lys2796Gln

missense

Exon 58

Damaging

Possibly damaging

 

c.8520 + 125 del C (Intron)

 

Frameshift Deletion

Exon 58

  

Wu p019

c.575G > A

p.Arg192Gln

missense

Exon 5

Tolerated

Benign

 

c.1422_1422delC

p.Lys476fs

Frameshift Deletion

Exon 13

  

Wu p021

c.1080_1083delAAGT

p.Lys362fs

Frameshift Deletion

Exon 10

  

Wu p022

c.1062G > C

p.Lys354Asn

missense

Exon 9

Tolerated

Possibly damaging

Wu p023

c.1062G > C

p.Lys354Asn

missense

Exon 9

Tolerated

Possibly damaging

Wu p024

c.1658A > C

p.His553Pro

missense

Exon 15

D

Possibly damaging

Wu p025

c.4316 T > A

p.Leu1439Ter

nonsense

Exon 32

Tolerated

 

Wu p027

c.1754_1757delTAAC

p.Thr586fs

Frameshift Deletion

Exon 16

  

Wu p030

c.5665G > T

p.Glu1889Ter

nonsense

Exon 39

Tolerated

 

Wu p032

c.2266C > T

p.Gln756Ter

nonsense

Exon 19

Damaging

 

Wu p033

c.7348C > T

p.Arg2450Ter

nonsense

Exon 50

Tolerated

 

Wu p034

c.910C > T

p.Arg304Ter

nonsense

Exon 9

Tolerated

 

Wu p035

c.5580_5581insA

p.Asn1861fs

Frameshift Insertion

Exon 38

  

Wu p038

c.1246C > T

p.Arg416Ter

nonsense

Exon 11

Tolerated

 

Wu p039

c.492_495 del AACT/He

p.Val166fs

Frameshift Deletion

Exon 5

  

Wu p041

c.910C > T

p.Arg304Ter

nonsense

Exon 9

Tolerated

 

Wu p043

c. 3796 G > T

p.Glu1266Ter

nonsense

Exon28

Tolerated

 

Wu p044

c.86_87delAC

p.29_29del

frameshift deletion

Exon2

  

Wu p045

c.6618_6618 delA

p.Thr2206fs

frameshift deletion

Exon43

  

Wu p047

c. 6818 A > C

p.Lys2273 Thr

missense

Exon46

Tolerated

Possibly damaging

Wu p048

c. 910 C > T

p.Arg304Ter

nonsense

Exon9

Tolerated

 

Wu p050

c.2212dupT

p.Phe738fs

frameshift insertion

Exon18

  

Wu p051

c. 5170 C > T

p.Gln1724Ter

nonsense

Exon37

Tolerated

 

Wu p052

c. 1224 T > A

p.Tyr408Ter

nonsense

Exon11

Tolerated

 

Wu p053

c.7266_7267del AC

p.2422_2423del

frameshift deletion

Exon49

  

Wu p054

c. 574 C > T

p.Arg192Ter

nonsense

Exon5

Tolerated

 

Wu p055

c. 574 C > T

p.Arg192Ter

nonsense

Exon5

Tolerated

 

Wu p058

c. 3040 A > T

p.K1014Ter

nonsense

Exon23

  

Wu p059

c.288 + 1G > T

.

splicing

Exon3

  

Wu p060

c.4509dupT

p.Asn1503fs

frameshift insertion

Exon34

  

Wu p064

c. 479 G > T

p.Arg160Met

missense

Exon4

Damaging

Possibly damaging

Wu p066

c.1592delA

p.Gln531fs

frameshift deletion

Exon14

  

Wu p067

c.8070dupC

p.Tyr2690fs

frameshift insertion

Exon56

  

Wu p068

c.288 + 1G > T

.

splicing

Exon3

  

Wu p070

c.4990_4992AAA (GTT)

.

nonframeshift substitution

Exon37

  

Wu p071

c. 3826 C > T

p.Arg1276Ter

nonsense

Exon28

Tolerated

 

Wu p073

c.2340_2346delACATGCA

p.780_782del

frameshift deletion

Exon20

  

Wu p074

c. 4107 C > A

p.Tyr1369Ter

nonsense

Exon30

Tolerated

 

Wu p075

c. 5651 T > G

p.Phe1884Cys

missense

Exon39

Damaging

Damaging

Wu p076

c. 3888 T > G

p.Tyr1296Ter

nonsense

Exon29

Tolerated

 

Wu p077

c. 3484 A > G

p.Met1162Val

missense

Exon26

Tolerated

Benign

Wu p077

c. 7189 G > A

p.Gly2397Arg

missense

Exon49

Damaging

Damaging

Wu p080

c. 1933 A > G

p.Met645Val

missense

Exon17

Tolerated

Benign

Wu p081

c.1754_1757del

p.Leu585fs

frameshift deletion

Exon16

  

Wu p083

c.2953dupC

p.Gly984fs

frameshift insertion

Exon22

  

Wu p086

c.6855C > A

p.Tyr2285Ter

nonsense

Exon46

Tolerated

 

Wu p087

c. 4940 A > C

p.His1647Pro

missense

Exon37

Tolerated

Damaging

Wu p088

c.1754_1757del

p.Leu585fs

frameshift deletion

Exon16

  

Wu p089

c. 1466 A > G

p.Tyr489Cys

missense

Exon13

Tolerated

Damaging

Wu p090

c. 376 G > T

p.Glu126Ter

nonsense

Exon4

Damaging

 

Wu p092

c. 3827 G > A

p.Arg1276Gln

missense

Exon28

 

Damaging

Wu p094

c. 3796 G > T

p.Glu1266Ter

nonsense

Exon28

Tolerated

Damaging

Wu p095

c.1693dupG

p.Asp564fs

frameshift insertion

Exon15

  

Wu p098

c.1754_1757del

p.Leu585fs

frameshift deletion

Exon16

  

Wu p100

c. 1318 C > T

p.Arg440Ter

nonsense

Exon12

Tolerated

 

abold lettering indicated as novel variants

Genetic alterations in the NF1 gene were detected as frameshift, nonsense, splice, missense mutations, and frame deletions or duplications from the first NGS panel screening (Fig. 1). These variants distributed along the NF1 gene without any clustering hotspot domain. Intragenic NF1 point mutations were found in 46 patients, 28 nonsense and 18 missense mutations. Small insertions and/or deletions were identified in 24 patients and most of them with frameshift consequences. Splice alterations were detected only in three patients. Four patients (Wu p003, Wu p018, Wu p019, and Wu p077) possessed more than one NF1 variant. Two patients with BRAF variants (c.74C > T in Exon1: p.Pro25Leu; c.G316A in Exon 3: p.Gly106Arg) were identified from this NGS screening. Both these patients also carried NF1 mutations (Fig. 2). On comparing with the Human Gene Mutation Database (HGMD, Institute of Medical Genetics, Cardiff, http://www.hgmd.org/), and Leiden Open Variation Database (LOVD: www.lovd.nl/NF1), we found that 48 variants of NF1 gene and two of BRAF gene are supposed to be novel (presented in bold in Table 2 and Table 3). All these novel mutations in this study were tested in 100 normal alleles.
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

Some represented results of Sanger sequencing at the mutation site with blood sample

Table 3

NF1 multi-exon deletions or duplications

NAME

MLPA

Clinical features

Tumor type

Wu p008

3’ UTR del/He

café-au-lait spot

Multiple cutaneous tumor

Wu p013

Exon 10 ~  58 del/He

café-au-lait spot

whole body

Wu p016

Exon 1 ~  58 del/He

café-au-lait spot, skin nodules

Two nodules of tumor involving the dermis and composed of spindle cells with wavy elongated nuclei

Wu p020

Exon 28~ 39 del/He

café-au-lait spot

Neurofibroma

Wu p029

Exon 4C~ 6 (no Exon 5)

café-au-lait spot, skin nodules

Right facial plexiform neurofibroma

Wu p031

Exon 1B~ 49

café-au-lait spot, skin nodules

multiple nodules over face and bilateral forearms

Wu p037

Exon1~ 58 del/He

café-au-lait spot/List Nodules in the Iris

multiple nodules over face

Wu p061

Exon37~ 51 del/He

café-au-lait spot

NF1 with optic nerve glioma

Wu p062

Exon2~ 8 del/He

café-au-lait spot/List Nodules in the Iris

right thigh subcutaneous layer soft tissue nodule

Wu p065

Exon 28–29 del/He

café-au-lait spot

lower limb plexiform NF

Wu p069

Exon1~ 58 del/He

café-au-lait spot/List Nodules in the Iris

Neurofibroma over back

Wu p082

Exon2~ 5 del/He

café-au-lait spot

plexiform neurofibroma over buttock

Wu p085

Exon2~ 5 del/He

café-au-lait spot

left optic nerve glioma & liposarcoma

Wu p093

Exon 1B ~ 4B

skin nodules/List Nodules in the Iris

Plexiform Neurofibroma

Wu p099

Exon 4C~ 6 (no Exon 5)

café-au-lait spot, skin nodules

skin and soft tissue on right face, plexiform neurofibroma

Spectrum of NF1 mutations identified by MLPA analysis

For patients who showed no detected mutations by our NGS panel screening, we then analyzed possible exons deletion/duplication within the NF1 gene using multiplex ligation-dependent probe amplification (MLPA) approach. Whole NF1 gene deletions were found in three patients and fifteen multi-exon deletions within the NF1 gene were obtained in this cohort of NF1 patients. Most of these exon deletions were only seen once in this study (Fig. 3, Table 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

Clinical features of NF1 patients with concurrence of NF1-BRAF mutations

Patient (Wu p001) was diagnosed as NF1 at the age of seven years by the presence of left craniofacial plexiform neurofibromas, infiltrative at the left temporal scalp, nodular subcutaneous tissue of the cheek, masticator space and probably the parotid space. He had multiple café-au-lait spots but no Lisch nodules. His brain magnetic resonance image (MRI) showed multiple unknown bright objects at pons, bilateral cerebellar hemisphere, globus pallidus and right thalamus. His left temporal and zygomatic bone showed progressive enlargement. His father (Wu p039) was the first patient with NF1 in this family and was diagnosed as having NF1 because of the presence of Lisch nodules, skeletal dysplasia, hundreds of café-au-lait spots and cutaneous nodular neurofibromas all over the body. This father and son are both intellectually normal. They both share the same genetic alterations on NF1 (c.492_495 del AACT/p.Val166fs) and BRAF (c.74C > T/p.Pro25Leu) gene (Fig.2). Another patient (Wu p083) was diagnosed as having NF1 at age of five years. He had multiple café-au-lait spots and Lisch nodules with soft tissue mass over the right back. He also had unspecified heart anomaly and T-spine scoliosis. His brain MRI showed white matter hyperintensity suggesting spongiform change at the left globus pallidus, dorsal pons, and bilateral cerebellar hemisphere (dentate nuclei) but no definite evidence of optic gliomas. He was detected as having NF1 (c.2953dupC/p.Gly984fs) and BRAF (c.316 G > A/p.Gly106Arg) genetic variants in the first NGS screening (Fig. 2, Table 2). These three patients presented no data for their definite atrial septal defect, ventricular septal defect and patent ductus arteriosus. In addition, none of these patients show the typical features of NF1–Noonan syndrome, Noonan syndrome or CFC syndrome.

Discussion

We here assessed a DNA-based approach combining targeted gene panel screening with MLPA analysis in a cohort of clinically suspected NF1 patients. On targeted gene panel screening, we identified 73 NF1 mutations and two BRAF variants (c.74C > T: p.Pro25Leu; c.316 G > A: p.Gly106Arg) in a total of 100 NF1 patients from 95 families diagnosed as having NF1 on the basis of the clinical criteria. These mutations are heterogeneous and distribute without clustering in either cysteine–serine-rich domain or within the GAP-related domain. 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, 16, 17, 18, 19, 20, 21, 26, 27, 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. Our analysis and this study may have missed the genetic variants residing in the promotor and intronic untranscribed non-coding regions or those involved in large genomic rearrangements or epigenetic mechanisms. We anticipate that whole-genome analysis may provide further insights for the information related to this issue.

NF1 is a progressive disorder complicated by the variability of disease expression. Beyond the primary concern of cutaneous/dermal neurofibromas, pigmented lesions, and the occasional limb abnormalities, the majority of NF1 patients do not fulfill the NIH criteria. Only ~ 30% of NF1 patients develop clinically detectable plexiform neurofibromas, and many features of NF1 only display café-au-lait spots and mild symptoms or no major disease complications in their early life [5, 36, 37]. Although neurofibromatosis type 1 is the most common syndrome seen in children with multiple café-au-lait spots, other syndromes associated with one or more café-au-lait spots include McCune-Albright syndrome, Legius syndrome, Noonan syndrome and other neuro-cardio-facio-cutaneous syndromes [38]. It also shares some features including reduced growth, facial dysmorphia, cardiac defects, skeletal and ectodermal anomalies, variable cognitive deficits, and susceptibility to certain malignancies with a group of clinically distinct developmental disorders [23, 24, 25]. Neurofibromatosis type I, Noonan syndrome, LEOPARD syndrome, and cardiofaciocutaneous syndromes were usually grouped as “neuro-cardio-facio-cutaneous” (NCFC) syndromes but are now called as “RaSopathies”. All these disorders involve a common Ras–Raf–signaling pathway [39, 40, 41]. To our knowledge, germline KRAS mutations occasionally occur in Noonan (2–4%) [42, 43, 44, 45, 46] and CFC syndromes (< 2%) [43, 44, 45, 47, 49]. Germline BRAF mutations can cause CFC syndrome (approximately 50–75%) [44, 47, 48, 49, 50], Noonan syndrome [47, 50], and LEOPARD syndrome type 3 (< 2%) [50, 51]. However, these individuals usually are not associated with neurofibromas (Table 4).
Table 4

BRAF mutations in patients with RASopathies

Patient

Germline mutation

Clinical Phenotypes

Tumor type

Wu p001 (this study)

NF1 Exon 5,

c.492_495 del AACT/p.Val166fs

Café-au-lait spots, Cutaneous neurofibroma, left zygoms progressive enlargement

plexiform neurofibroma

 

BRAF Exon 1, c.74C > T/p.Pro25Leu

  

Wu p083 (this study)

NF1 Exon22, c.2953dupC/p.Gly984fs

Café-au-lait spots, unspecified cardiac anomaly, Lisch Nodules in the Iris, T-spine scoliosis

paraspinal plexiform neurofibroma

 

BRAF Exon 3, c. 316 G > A/p.Gly106Arg

  

Noonan syndrome (NS)

BRAF (T241 M; T241R; W531C; L597 V)

Short stature, dysmorphic facial features, mild-to-moderate cognitive deficits, skeletal anomalies, and hypotonia

 

Cardio-facio-cutaneous syndrome (CFCS)

BRAF (L245F; A246P; T241P; Q257R; G469E; etc)

Dysmorphic facies, cardiac defects, and skin and skeletal anomalies

 

Leopard syndrome Type 3

BRAF (T241P; L245F)

Craniofacial anomalies, short and webbed neck, cardiac conduction defects, Multiple pigmented skin lesions and showed growth retardation, delayed puberty, and delayed bone age.

undetected

*bold lettering indicated as novel variants

Phenotypic variation could result from different expression patterns of mutated genes, as well as from different mechanisms that disturb RAS signaling through specific interactions with effector and regulatory proteins for different mutants. Variability could also result from the feedback mechanisms that can affect upstream molecules (like RAS) but not downstream molecules [40]. Therefore, a NGS panel with high coverage of Ras–signaling components should be very useful in clinical diagnosis. However, we cannot yet explain how the concurrence of NF1 and BRAF variants contributes to NF1 in these patients.

Conclusion

Differential diagnosis of NF1-like patients is still challenging owing to its clinical complexity. A genetic screening using a NGS panel in high coverage of Ras–signaling components combined with Multiple Ligation-Dependent Probe Amplification analysis should enable us to get the molecular control of these clinically overlapping disorders. We believe that the availability of whole-genome analysis will provide an opportunity for the genetic diagnosis of NF1 and will bring more insights for the development of NF1.

Notes

Acknowledgements

We are grateful to all the participating patients and their families who made these studies possible. The authors also thank the DNA Sequencing Core Laboratory at the Chang Gung Memorial Hospital, Linko.

Funding

Dr. Wu-Chou received funding by grants from the National Science Council, Taiwan (NSC94–2320-B-182A-020) and the Chang Gung Research Foundation (CMRPG 33139, CMRPG 331393, CMRPG 3D0391–3).

Availability of data and materials

The datasets generated in the current study are available from the corresponding author on request.

Authors’ contributions

YHWC conceived and designed the study, carried out the lab data analysis, interpreted the results and drafted the manuscript. The study subjects were assessed by the pediatric physician JLL, neurological physician THY, and plastic surgeons CHL, CCY, KTC, and YRC at the Chang Gung Craniofacial Center. YTL contributed to participant recruitment, acquisition of samples and experimental data. ZCH and HWC performed molecular genetic experiments. All authors have approved the final manuscript for submission.

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.

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© The Author(s). 2018

Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.

Authors and Affiliations

  • Yah-Huei Wu-Chou
    • 1
  • Tzu-Chao Hung
    • 1
  • Yin-Ting Lin
    • 1
  • Hsing-Wen Cheng
    • 1
  • Ju-Li Lin
    • 2
  • Chih-Hung Lin
    • 3
  • Chung-Chih Yu
    • 3
  • Kuo-Ting Chen
    • 3
  • Tu-Hsueh Yeh
    • 4
  • Yu-Ray Chen
    • 3
  1. 1.Human Molecular Genetics Laboratory, Department of Medical ResearchChang Gung Memorial HospitalTaoyuanTaiwan
  2. 2.Division of Genetics and Endocrinology, Department of PediatricsChang Gung University College of Medicine and Chang Gung Children’s and Memorial HospitalTaoyuanTaiwan
  3. 3.Department of Plastic & Reconstructive SurgeryChang Gung Memorial HospitalTaoyuanTaiwan
  4. 4.Neuroscience Research Center, Department of NeurologyChang Gung Memorial HospitalTaoyuanTaiwan

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