SHORT COMMUNICATION

Familial spinal neurofibromatosis due to a multiexonic NF1

gene deletion

Antonio Pizzuti & Irene Bottillo & Francesca Inzana & Valentina Lanari &

Francesca Buttarelli & Isabella Torrente & Anna Teresa Giallonardo &

Alessandro De Luca & Bruno Dallapiccola

Received: 21 September 2010 /Accepted: 4 February 2011# Springer-Verlag 2011

Abstract We report the detailed clinical presentation and

molecular features of a spinal neurofibromatosis familial

case where a 40-year-old woman, presenting with multiple

bilateral spinal neurofibromas and no other clinical feature

of neurofibromatosis type 1 (NF1), inherited a paternal

large multiexonic deletion (c.5944−?_7126+?del) which

resulted in NF1 gene haploinsufficiency at the RNA level.

In the clinically unaffected 73-year-old father, spinal cord

MRI disclosed bilateral and symmetrical hypertrophy of

spinal lumbosacral roots. Our study widens the phenotypic

and mutational spectrum of NF1 and illustrates the

difficulties of counseling patients with border-line or

atypical presentation of this disorder.

Keywords FSNF. HSNF.NF1 . Spinal neurofibromas .

Neurofibromatosis type 1

Introduction

Neurofibromas are benign tumors of the peripheral nerves,

typically found in individuals with neurofibromatosis type

1 (NF1, MIM 162200), a genetically inherited disease.

These lesions arise from Schwann cells exhibiting biallelic

inactivation of the NF1 gene, located at 17q11.2, which

codes for neurofibromin [1, 2], an important negative

regulator of the cellular Ras signaling pathway [3–6].

Dermal neurofibromas are well-circumscribed solid

cutaneous tumors with limited size. They look like lumps

on or under the skin, but may also arise from dorsal nerve

roots [7]. These spinal tumors are symptomatic (usually

with back pain and sensory deficit in the limbs) in only 5%

of NF1 patients [8], although they can be observed by

magnetic resonance imaging (MRI) in up to 36% of them

[9–11].

The presence of a wide, bilateral distribution of multiple

spinal neurofibromas symmetrically at the cranial, thoracic,

and lumbar vertebral level, occurring in adult members of

the same family and segregating with an autosomal

dominant fashion, is extremely rare [12, 13]. This form of

disease, named familial spinal NF (FSNF, MIM 162210)

[10, 14–16] or hereditary spinal neurofibromatosis [10], has

been regarded as an alternative form of NF1 since patients

generally lack dermal neurofibromas and Lisch nodules,

both hallmarks of NF1, and, on the other hand, symptom-

atic generalized spinal neurofibromas are uncommon in

classical NF1. A number of FSNF families have been

reported, and NF1 gene mutations are characterized [10,

16–23]. The NF1 locus was not involved only in one of the

A. Pizzuti

Department of Experimental Medicine, “Sapienza” University,

Rome, Italy

I. Bottillo : F. Inzana :V. Lanari : I. Torrente :A. De Luca

IRCCS Casa Sollievo della Sofferenza, Mendel Laboratory,

San Giovanni Rotondo, Italy

F. Buttarelli

Department of Neurology and Psychiatry, “Sapienza” University,

Rome, Italy

A. T. Giallonardo

Department of Neurological Sciences,

University of Rome “Sapienza”,

Rome, Italy

B. Dallapiccola

Bambino Gesù Children Hospital, IRCCS,

Rome, Italy

B. Dallapiccola (*)

Ospedale Bambino Gesù, IRCCS,

Piazza S. Onofrio 4,

00165 Rome, Italy

e-mail: bruno.dallapiccola@opbg.net

Neurogenetics

DOI 10.1007/s10048-011-0278-5

investigated cases [16]. The NF1 germline mutational

spectrum consists of a high incidence of truncating

mutations [20] both in patients affected by typical NF1

and spinal neurofibromas and the general NF1 population,

while in cases with multiple spinal neurofibromas but very

few or no other clinical signs of NF1, a considerable

number of missense mutations and splicing mutations has

been reported [20, 24].

To determine whether the genotype may be a major

determinant for FSNF development and identify those NF1

gene alterations that eventually predispose to this relatively

mild form of NF1, it is important to genetically characterize

and report the largest possible number of families transmitting

this trait.

Here, we provide detailed clinical presentation and

molecular description of a FSNF family in which an adult

woman presenting only with multiple symptomatic spinal

neurofibromas and no other NF1 clinical feature, inherited

from the apparently unaffected father a large multiexonic

deletion resulting in NF1 gene haploinsufficiency at the

RNA level.

Materials and methods

Patients

This study concerns the NF1 gene analysis of family FSNF-1

(Fig. 1a). The family presented with four individuals in two

generations. The project was approved by the institutional

review board, and all participants provided informed

consent. Peripheral blood was collected and stabilized both

by EDTA containing tubes and PAXgene Blood RNA

System tubes (PreAnalytiX, Hombrechtikon, Switzerland).

DNA was extracted by use of the Maxwell 16 DNA

Purification Kit (Promega, Madison, WI), while RNA was

isolated by the use of PAXgene Blood RNA kit (Qiagen,

Hilden, Germany). DNA extraction from paraffin-embedded

spinal tumor samples was as previously described [25].

Mutation analysis

Genomic DNA was analyzed to determine NF1 mutations.

Identification of point mutations was carried out by

denaturing high performance liquid chromatography

(dHPLC) followed by direct sequencing as described

elsewhere [26]. Forward and reverse sequences were

analyzed and compared with the mRNA reference sequence

(NM_000267). The exons are not named consecutively but

according to the accepted nomenclature used by researchers

in the NF1 field [27]. Screening for NF1 single- and

multiexonic copy number changes was carried out using the

SALSA P081/082 NF1 (version 04, 05-02-2005) MLPA

assay (MRC Holland, Amsterdam, The Netherlands) as

previously described [28]. With this method, the results

have been given as allele copy numbers while compared to

normal controls and a ratio of ~1 should be obtained if

both alleles are present. A decrease or increase in the peak

area values of <0.7 or >1.3 was considered an indication

of either a deletion or a duplication, respectively. Each

variant identified with MLPA analysis was confirmed by a

real-time based on SYBR green assay [28] (data not

shown).

Total RNA of patients I:1, I:2, and II:2 was isolated by

the use of PAXgene Blood RNA kit (Qiagen, Hilden,

Germany) from peripheral blood lymphocytes and collected

and stabilized by PAXgene Blood RNA System tubes

(Qiagen, Hilden, Germany). First strand cDNA was

synthesized starting from 2 μg of total RNA by random-

primed reverse transcription with Superscript II Reverse

Transcriptase (Invitrogen Corporation, Carlsbad, CA). The

following primers were used for cDNA-PCR amplification:

FW: 5′-ATGACCATCAATGAAAAACAGATG-3′ (located

in NF1 exon 31) and Rv: 5′-AGCCTTTGTGTCTGATAT

CAAG-3′ (located in NF1 exon 43). The PCR amplification

reaction was performed as follows: in 25 μL volume, 2 μl

of cDNA, 0.2 μM primers, 100 μM dNTP, 5 μl reaction

Gold buffer (Applied Biosystems, Foster City, CA), 2 mM

MgCl2, and 2.5 U AmpliTaq Gold Polymerase (Applied

Biosystems, Foster City, CA), in a 9700 (Applied Biosystems,

Foster City, CA, USA) thermal cycler. Thermal conditions

were 35 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for

2 min, preceded by 2 min at 95°C and followed by a final

elongation step at 72°C for 10 min. The entire PCR volume

(25 μl) was run on a 2% agarose gel.

The occurrence of loss of heterozygosity (LOH) encom-

passing NF1 region was studied using the battery of

microsatellite markers flanking or intragenic to the NF1

gene [29]. For heterozygous samples, a reduction of at least

50% in the peak height of one allele in the tumor was used

to score LOH [26].

Results

The proband (Fig. 1a, II:2) was the second daughter of non-

consanguineous parents. We firstly evaluated her at the age

of 29 for neurological symptoms consisting in sensory

deficit and strength reduction in the left limbs, especially

the upper limb. She presented gait disturbance and

weakness at lower limbs, with sudden movements causing

falls. She complained pain in the right lower limb and pin-

point pain of the left part of the skull, sparing only the face.

Neurological evaluation showed deficit of the deep sensi-

bilities on the left side. MRI disclosed enlarged spinal nerve

roots from C4 to C6, with suppression of the epidural fat

Neurogenetics

sign around them, interpreted as multiple roots tumors. A

tumor of T10 root was also found. The proband was

initially treated by physiotherapy with partial symptoms

remission. An additional neurological assessment revealed

spastic paraparesis and hyperreflexia in the lower limbs. At

the age of 34, the patient underwent surgery for removal of

five intraforaminal spinal tumors at roots C4–C5 and C5–

C6. Histological examination was consistent with a diag-

nosis of neurofibromas. Following surgery, the patient

showed a marked improvement in spasticity and pain, but

a slight walking difficulty remained, with a wide base gait,

and pain in the left upper limb, especially in the hand,

accompanied by hypoesthesia. An additional MRI scan at

the age of 35 showed the same alterations extended at roots

C1–C2, from C3 to C6, and at lumbosacral level. After

1 year, another MRI demonstrated that neurofibromas were

present in all spinal roots, bilaterally and symmetrically.

Neurological evaluation documented spasticity at all limbs.

In particular, spasticity was prevalent in the right lower

limb and strength deficit in the left one, while pain involved

Fig. 1 a Pedigree of the FSNF family. Individuals harboring

(c. 5944−?_7126+?del) NF1 gene mutation are denoted by a

blackened symbol. b P081–P082 MLPA results. Normalized relative

peak areas of all NF1 gene-specific and control probes are shown.

Sequences present in two copies of the genome have a relative peak

area value of approximately 1.0 (white histograms). A reduction in

the peak area values to 0.7 indicates a deletion (gray histograms).

Patients I:1 and II:2 carry a deletion spanning from NF1 exon 32 to

exon 39 (corresponding to exon 41 and 48 according to the

nomenclature now recommended through NCBI and HGVS), while

individuals I:2 and II:1 and II:4 have a wild-type genotype. c Quantitative

PCR assay results to assess the copy number state of NF1 exon 37;

x-axis and y-axis represent analyzed DNA samples and N-fold values,

respectively. Sample c1 is a deleted positive control. Samples c2 and c3

are two non-deleted negative controls. Gray histograms represent N-fold

values corresponding to two copies of the test NF1 exon. Black

histograms represent N-fold values corresponding to one copy of the

test NF1 exon. d cDNA-PCR products amplified from individuals I:1,

I:2, and II:2 lymphocyte RNA and run on a 2% agarose gel. The single

band indicates a 1,719-bp product corresponding to the only expression

of the wild-type allele

Neurogenetics

both sides. Two years later, an additional MRI confirmed

the spinal picture of the previous scans and revealed a

medullary compression at cervical level (C3–C5). At the

age of 40, the patient underwent another MRI which

disclosed the same alterations previously found and no

cerebral tumors (Fig. 2). She went through genetic

counselling. The family history for NF1 or other genetic

diseases was negative and she did not show any cafe-au-lait

spots, cutaneous neurofibromas, Lisch nodules, freckling,

or any other feature of NF1. The significant spinal

involvement in the absence of other signs of NF1 was

compatible with the clinical diagnosis of sporadic spinal

neurofibromatosis, and therefore, after blood drawing,

molecular analysis of NF1 gene was carried out. While

dHPLC mutation analysis did not identify any nucleotide

chance, MLPA analysis of the NF1 gene revealed the

presence of a multiexonic deletion (c. 5944−?_7126+?del)

encompassing eight NF1 exons (extending from exon 32 to

Fig. 2 Patient II:2 spinal MRI.

a, b Cervical tract (T2-weighted,

sagittal scan): cervical neurofi-

bromas, symmetrically and bilat-

erally distributed. c, d Cervical

tract (T1-weighted, axial scan):

neurofibromas of the spinal

roots, enlarging vertebral forami-

na. Note the absence of the

epidural fat around roots. e, f

Lumbar and sacral tracts

(T1-weighted, sagittal scan):

neurofibromas of the

lumbosacral spinal roots

Neurogenetics

exon 39 according to the accepted nomenclature used by

researchers in the NF1 field [27], and corresponding to

exon 41 to 48 according to the nomenclature recommen-

ded by NCBI, http://www.ncbi.nlm.nih.gov/ and HGVS,

http://www.hgvs.org/rec.html) (Fig. 1b). Presence of

(c. 5944−?_7126+?del) mutation in individual II:2 was

confirmed using a SYBR green-based real-time quantitative

PCR assay (Fig. 1c). To confirm the supposed sporadic

nature of the identified variant, we tested parental

lymphocytes DNA (individuals I:1 and I:2). The asymp-

tomatic father (individual I:1) was found to carry the same

mutation. He was a 73-year-old man with no clinical

feature of classical NF1 even after an accurate clinical

examination. At the same time, he underwent spinal cord

MRI. This investigation disclosed the presence of hyper-

trophy of spinal lumbosacral roots on both sides, with

symmetrical distribution (Fig. 3). Table 1 summarizes the

clinical findings of individuals I:1 and II:2. To study the

effect of the mutation on the NF1 transcript, lymphocyte

RNA from the affected individuals I:1 and II:2, and from

the unaffected mother I:2, was reverse transcribed and

analyzed by reverse transcription PCR. A single band of

1,719 bp corresponding to the expression of the wild-type

allele only was detected in all analyzed individuals

(Fig. 1d). Thus, the expression of the mutant allele must

be either completely abolished or very low. This result was

confirmed by sequence analysis (data not shown).

One spinal tumor from patient II:2 was available as

paraffin-embedded block for LOH analysis. Four micro-

satellite loci (D17S1873, D17S635, D17S1166, 3'NF1-1)

were informative for evaluation of LOH. By the criteria we

employed, LOH was not detected at any microsatellite

examined in the original tumour specimen (data not shown).

Discussion

Spinal neurofibromas occur in 36–40% of classic NF1

individuals but yields neurological symptoms in no more

than 2–5% of the patients [9, 11, 30, 31]. Conversely,

symptomatic multiple spinal neurofibromas are the major

clinical features of FSNF [14, 23]. This condition has been

considered an alternative form of NF1 because of the

exceptional occurrence of multiple spinal neurofibromas

affecting spinal roots in all affected adult members of the

same family as well as very mild cutaneous signs of NF1

and absence of Lisch nodules [12]. These distinct clinical

Fig. 3 Patient I:1 spinal MRI of

the lumbosacral spinal tract

(T1-weighted, sagittal scan):

neurofibromas of the lumbosacral

roots, bilaterally and

symmetrically distributed

Table 1 Clinical features of individuals with FSNF

Patient II:2 I:1

Sex F M

Age of onset 29 ?

Age at evaluation 40 73

CLS − −

CNF − −

Lisch nodules − −

Freckling − −

Scoliosis − −

PNF − −

Spinal MRI Multiple NFs Multiple NFs

Roots involved All L/S

Symmetry + +

Symptoms + −

F female, M male, + present, −absent, ? unknown, CLS cafè-au lait

spots, CNF cutaneous neurofibromas, PNF plexiform neurofibromas,

MRI magnetic resonance imaging, NF neurofibromas, L lumbar, S sacral

Neurogenetics

findings have suggested that a genotype–phenotype corre-

lation might be found in these families and that a specific

type of gene defect with a special effect on neural crest cells

and/or their precursors in the nerve roots might be present

in these families. To our knowledge, including the present

study, 12 families and 7 sporadic patients with FSNF have

been described at molecular level so far [10, 14, 16, 17, 19–

23]. Of these, 6/12 (50%) families and 1/7 (14%) sporadic

patients harbor a truncating mutation [16, 17, 19, 20, 23], 5/

12 (42%) families and 4/7 (57%) sporadic patients harbor a

missense mutation [10, 14, 19–22], and 1/12 (8%) families

[present study] and 2/7 (29%) sporadic patients harbor a

partial or whole gene deletion [22]. Therefore, differently

from classic NF1, in which the prevalence of missense

changes is rather rare (28/278, 10%) [32], a quite high

incidence (9/19, 50%) of missense NF1 gene mutations

seems to occur in patients with FSNF (Fisher's exact test,

p=0.001).

Based on the observation that many of the mutations

found in FSNF are missense and splicing variants, or

truncating mutations mapping at the 3′ end of the NF1

gene, it has been hypothesize that FSNF patients might

harbor “mild” NF1 mutations leading to some neuro-

fibromin residual function associated with different con-

sequences on the development of neural crest cells [14, 17].

Even though it is possible that specific NF1 alterations

found in FSNF patients might have a “milder” effect on

protein function, it is unlikely that the multiexonic deletion

detected in our FSNF family encodes for a protein with

some residual activity. Although the deletion was located

downstream to the catalytic neurofibromin domain (GAP-

related domain), the RNA-based study performed on

peripheral blood lymphocytes clearly showed that the

deleted allele results in loss or very low expression of the

NF1 gene. Although MLPA technique does not map

precisely the deletion breakpoints, it is likely that they

reside within introns 31 and 39. A deletion comprising NF1

exons 32–39 is predicted to get rid of 1,183 nucleotides

from the coding sequence. The resulting protein would be

an aberrant neurofibromin correctly encoded till tyrosine

residue 2,377, after which the protein would be frame-

shifted for 19 amino acids and then interrupted because of

the introduction of a premature termination codon. Such

protein is supposed to be degraded by the nonsense-

mediated mRNA decay, a mechanism proven to influence

the stability of NF1 transcripts encoding for truncated

proteins [33]. This hypothesis is in agreement with the

RNA findings (Fig. 1d). Recently, two sporadic patients

with spinal neurofibromas, both exhibiting a mild clinical

phenotype, were found to carry a large deletion encompass-

ing the entire NF1 gene. It has been supposed that they

might have been affected by a mosaicism restricted to the

spinal nerve roots [22]. However, the presence of only one

or two spinal tumors in these patients was indicative that

they differed from most FSNF cases, including the present

family, in which multiple, symmetrical spinal tumors are

typically found [22].

LOH is responsible for somatic inactivation of NF1

in ~20% of neurofibromas from individuals with a known

NF1 germline mutation [34, 35]. Here, we used a battery of

microsatellite markers to examine for LOH the NF1 locus

in a spinal tumor from a patient with FSNF. No evidence

for LOH was found in the examined tumor. Accordingly,

LOH was previously identified in 36% of the spinal tumors

from patients with FSNF [22]. It might be that the second

hit in the examined tumor is not represented by LOH but by

a point mutation, a mutational mechanism that has been

observed in spinal tumors [22].

The striking characteristic of the FSNF family reported

here is the significant spinal involvement in absence of any

other clinical manifestation of NF1. Interestingly, the

mutation positive, clinically asymptomatic heterozygous

father showed evidence of hypertrophic radiculopathy,

reminescent of the described cases of neurofibromatous

neuropathy [36]. The hypertrophic radiculopathy may well

be a “pre-neurofibromatous” pathological change confined

to the spinal roots.

The limited number of samples with FSNF reported so

far, and the considerable phenotypic variability of NF1

preclude drawing conclusions on whether specific NF1

mutations predispose to FSNF or whether NF1 gene clinical

expression is modified in these patients. However, some

NF1 mutations reported in patients with FSNF (IVS31-

5A>G [19], IVS19b-3C>G [23], 1.4 Mb entire gene

deletion [22]) have been also identified in individuals with

classic NF1 presentation [25, 26]. In these cases, it is likely

that the FSNF phenotype is the result of cooperation

between the NF1 mutation and mutations in other genes

(modifiers). Gene expression studies and mutation analyses

of candidate modifiers in families and in isolated patients

presenting with the NF1 spinal-specific phenotype will

shed more light on this hypothesis and will eventually help

to understand how these genes modulate the phenotypic

expression of NF1 gene. The present spinal NF1 family, in

which a relatively mild NF1 phenotype was associated to

the transmission of a putative loss of function mutation,

illustrates the difficulty in counseling NF1 patients with

border-line clinical features or atypical presentation. In

addition, we expect that it will help the discussion around

the molecular alterations that cause this distinct neurological

phenotype.

Acknowledgements This work was supported by the Italian

Ministry of Health, Ricerca Corrente 2010. All authors have read

and approved the manuscript, and they have disclosed any financial

conflict of interest that may be used for influencing the results.

Neurogenetics

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