REVIEW

Legius Syndrome, an Update.
Molecular Pathology of Mutations in SPRED1
Hilde Brems and Eric Legius

Department of Human Genetics, KU Leuven, Leuven, Belgium(Received for publication on April 13, 2013)(Revised for publication on July 30, 2013)(Accepted for publication on September 19, 2013)(Published online in advance on December 10, 2013)

Multiple café-au-lait macules (CALMs) are the hallmark of Von Recklinghausen disease, or neurofibromatosis type 1 (NF1). In 2007 we reported that some individuals with multiple CALMs have aheterozygous mutation in the SPRED1 gene and have NF1-like syndrome, or Legius syndrome. Individuals with Legius syndrome have multiple CALMs with or without freckling, but they do not show thetypical NF1-associated tumors such as neurofibromas or optic pathway gliomas. NF1-associated boneabnormalities and Lisch nodules are also not reported in patients with Legius syndrome. Consequently,
individuals with Legius syndrome require less intense medical surveillance than those with NF1. The
SPRED1 gene was identified in 2001 and codes for a protein that downregulates the RAS-mitogen activated protein kinase (RAS-MAPK) pathway; as does neurofibromin, the protein encoded by the NF1
gene. It is estimated that about 1–4% of individuals with multiple CALMs have a heterozygous SPRED1
mutation. Mutational and clinical data on 209 patients with Legius syndrome are tabulated in an online database (http://www.lovd.nl/SPRED1). Mice with homozygous knockout of the Spred1 gene showlearning deficits and decreased synaptic plasticity in hippocampal neurons similar to those seen in Nf1
heterozygous mice, underlining the importance of the RAS-MAPK pathway for learning and memory.
Recently, specific binding between neurofibromin and SPRED1 was demonstrated. SPRED1 seems toplay an important role in recruiting neurofibromin to the plasma membrane. (doi: 10.2302/kjm.20130002-RE; Keio J Med 62 (4) : 107–112, December 2013)

Keywords: Legius syndrome, neurofibromatosis type 1, SPRED1, NF1, café-au-lait macule

Introduction
Neurofibromatosis type 1 (NF1) is an autosomal dominant genetic disorder with an incidence of 1/3000 births.1
Typical manifestations include multiple café-au-lait
macules (CALMs), freckling, Lisch nodules on the iris,neurofibromas, and optic pathway gliomas. Other symptoms are learning disabilities, macrocephaly, and specificskeletal abnormalities. Individuals with NF1 have an increased risk for specific malignancies.2
NF1 is a member of the RASopathy syndromes. RASopathies are caused by germline mutations in genes cod-ing for proteins with an important role in the RAS-mitogen-activated protein kinase (RAS-MAPK) pathway.3
RASopathies exhibit overactivation of the RAS-MAPK
pathway. Examples of such syndromes include Noonan,
cardio-facio-cutaneous, LEOPARD, Costello, capillarymalformation–arteriovenous malformation, and CBLmutation associated syndromes. These syndromes havean overlapping phenotype, although specific features
can often be distinguished. In 2007 we identified a new
syndrome resembling NF1. This syndrome is caused byheterozygous germline mutations in SPRED1.4 Initiallythe syndrome was named Neurofibromatosis type 1-likePresented at the 1842nd Meeting of The Keio Medical Society in Tokyo, October 31, 2012.
Reprint requests to: Eric Legius,MD, PhD, Department of Human Genetics, KU Leuven, Herestraat 49, 3000 Leuven, Belgium,
E-mail: Eric.Legius@uzleuven.be
Copyright © 2013 by The Keio Journal of Medicine

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Brems H and Legius E: Legius Syndrome, an Update

Fig. 1 Schematic of the RAS/mitogen-activated protein kinase pathway.
Associated developmental syndromes (RASopathies) are indicated by dashed lines. RAS is inactive when attached to GDP and isactive when attached to GTP. The transition from inactive to active RAS is mediated by SOS1 and the transition from active RAS
to inactive RAS is mediated by neurofibromin and p120GAP. SPRED1 is a negative regulator of BRAF and CRAF activation by active RAS. CM-AVM, capillary malformation–arteriovenous malformation syndrome; CFC, cardio-facio-cutaneous syndrome; CBL,
Cas-Br-M (murine) ectropic retroviral transforming sequence; Loh syndrome is a Noonan-like syndrome with a high risk of juvenile
myelomonocytic leukemia.

syndrome, but later it was renamed Legius syndrome.5,6
Legius syndrome also belongs to the group of RASopathies (Fig. 1).
Clinical Features of Legius Syndrome
NF1-like syndrome (Legius syndrome) was initiallyreported in five families (37 individuals) and in six unrelated individuals.4 It is not possible to diagnose Legius
syndrome based on clinical features alone because of theimportant clinical overlap with NF1. Individuals with Legius syndrome show the same multiple CALMs with orwithout axillary or inguinal freckling as seen in NF1 (Fig.
2). However, in Legius syndrome, certain other NF1-associated features such as Lisch nodules, neurofibromas,
NF1-specific bone lesions, optic pathway gliomas, andmalignant peripheral nerve sheath tumors are systematically absent (Table 1). Additional clinical features havebeen reported in Legius syndrome: pectus excavatum or
carinatum 12/877–10 and unilateral postaxial polydactyly(3/159).7,9 Lipomas are present in some individuals with
Legius syndrome.4,7,9–11
It is not yet clear whether malignancies are associatedwith Legius syndrome. A small number of neoplastic
events have been reported only once in an individual with
Legius syndrome, i.e., monoblastic acute leukemia,11,12
giant cell tumor, dermoid tumor of the ovary, breast cancer,9 vestibular schwannoma, and desmoid tumor.7 No
second hit was found in SPRED1 in monoblastic acuteleukemia,12 and no mutations in SPRED1 were identified
in a set of juvenile myeloblastic leukemia cases.13
Learning disabilities, developmental delay, Noonanlike characteristics, Attention Deficit (Hyperactivity)
Disorde, hyperactivity, autistic behavior, and concentration problems are frequently reported in children with
Legius syndrome. A cognitive phenotype was suggested

Keio J Med 2013; 62 (4): 107–112

109

Fig. 2 Café-au-lait macules on the back of an adult with Legius syndrome.

Table 1 Comparison of clinical features of neurofibromatosis type 1 and Legius syndrome
Neurofibromatosis type 1

Legius syndrome

++
++
+
+/−
+/−
++
50%
+/−
+
++
+/−
+
+

++
++
+
+/−
+/−
+
>60%
−
−
−
−
−
−

Café-au-lait macules
Freckling
Macrocephaly
Noonan-like facial dysmorphy
Pulmonary valve stenosis
Learning disabilities
Familial occurrence
Juvenile xanthogranuloma
Lisch nodules
Neurofibromas
Malignant peripheral nerve sheath tumor
Optic pathway glioma
Bone lesions(sphenoid wing dysplasia or pseudarthrosis)

in 15 individuals with Legius syndrome.14 These children
showed a lower average performance IQ and more learning disabilities compared to unaffected family members.
Based on these few observations, we believe that a cognitive phenotype is present in Legius syndrome but it ismilder than that in NF1.
Because the total number of reported cases of Legius
syndrome is relative small (159, all ages), rare complications could have been missed. To detect a rare complication with a prevalence of 1%, at least 250 adults with
Legius syndrome should be investigated.9 Therefore it
is important to keep collecting detailed clinical information from newly diagnosed individuals with Legius
syndrome. It is estimated that about 1–4% of individuals with multiple CALMs and a clinical diagnosis of NF1
in fact have Legius syndrome caused by a heterozygous
SPRED1 mutation.15

SPRED1 Gene
The Sprouty-related, EVH1 domain containing 1(Spred1) gene was discovered in 2001 by Yoshimura andcolleagues in a mouse cDNA osteoclast library.16 Human SPRED1 is located on chromosome band 15q13.2
and spans 104.4 kb of genomic sequence. It consists ofseven exons. The transcript is 7255 bp in length with anopen reading frame coding for 444 amino acids. In 2007,
linkage analysis in two of five families with an NF1-likephenotype mapped the phenotype to a region on chromosome 15 where SPRED1 had been previously localized.
Inactivating germline SPRED1 mutations were detectedin the affected individuals of these five families and inan additional six unrelated patients with a similar phenotype.4 Molecular genetic testing for Legius syndrome canbe performed on DNA or RNA extracted from periph-

110Brems H and Legius E: Legius Syndrome, an Update

Fig. 3 Schematic of the seven exons of the human SPRED1 gene and their corresponding functional domains in the protein.
EVH-1, EVH-1 domain; KBD, c-KIT-binding domain; SPR, SPROUTY-related domain. Protein truncating mutations are listed above
the exons. *, stop codon; fs, frameshift mutation; SPL, splice mutation. Missense mutations and deletions are listed below the exons.
IC, missense mutation affecting initiation codon; P, proven pathogenic missense mutation; U, unclassified missense mutation; SP,suggested pathogenic missense mutation; B, benign sequence variant without effect on SPRED1 function; SNP, single nucleotide
polymorphism. The numbers in parentheses indicate the number of independent individuals reported with that specific mutation.

eral white blood cells.4,9 Copy number changes such as
multi-exon deletions and whole SPRED1 gene deletions
can be detected by several methods such as multiplex ligation-dependent probe amplification, array comparative
genomic hybridization, or other techniques that detect
deletions.10
All identified mutations and polymorphisms in
SPRED1 are summarized in the Leiden Open Variation
Database, which is accessible online at http://www.lovd.
nl/SPRED1.15 Most of these mutations are predicted to beprotein truncating. From the 29 missense mutations currently reported, 8 are probably rare benign variants, 3 areclearly pathogenic, and 1 was suggested to be pathogenic(based on amino acid conservation and segregation withina large family). Some missense mutations are still unclassified and need further investigation.15 Familial as well as
sporadic cases of Legius syndrome have been reported;the largest study described 13/33 (39%) sporadic cases in
the SPRED1-positive group,9 but it should be taken into
account that this study was biased toward sporadic cases.
Only one somatic mutation has been published so far.4

In that study by Brems et al., melanocytes were cultured
from a CALM from an individual with Legius syndrome.
A somatic frameshift SPRED1 mutation was identified inaddition to the known germline SPRED1 mutation. Thisshows that CALMs in individuals with Legius syndromeare caused by biallelic inactivation of the SPRED1 gene,a mechanism similar to the biallelic inactivation of the
NF1 gene found in melanocytes derived from CALMs of
individuals with NF1.17 An overview of the mutations reported in the SPRED1 gene can be found in Figure 3.
SPRED1 Protein
Human SPRED1 contains 444 amino acids. Threefunctional domains have been identified: an N-terminal
EVH-1 domain, a central c-KIT-binding domain, and a
C-terminal SPRY-related domain. The SPRED1 protein isapproximately 50 kDa in size and belongs to the SPRED
family together with SPRED2 and SPRED3 (reviewed by
Yoshimura18). Spred1 is a negative regulator of the RasMAPK signaling pathway.16 Spred1 is enriched in the

Keio J Med 2013; 62 (4): 107–112

central nervous system germinal zones, it downregulates
neural stem cell proliferation and maintains ventricularzone structure.19 Human SPRED1 is highly expressedin lung, brain, spinal cord, and spleen. Lower expression levels are seen in liver, pancreas, prostate, kidney,
heart, thymus, muscle, and bone marrow.20,21 Spred1 is
phosphorylated by kinases and regulates activation of the
Ras-MAPK cascade in response to several growth factors without affecting the Akt or Rac pathway.16 Spred1
inhibits the activation of Raf by active Ras (Ras-GTP).16
Several Spred1 interaction partners have been identified, i.e., microtubule-associated protein/microtubule
affinity-regulating kinase-activating kinase (MARKK),
testis-specific protein kinase (TESK1),22 fibroblast
growth factor receptor like-1 (FGFRL1),23 DYRK1A,24
and SHP2.25 Recent research has shown that Spred1 binds
to neurofibromin and recruits it to the cell membrane
where neurofibromin can function as a negative regulator of Ras-GTP.26 It has been shown that some SPRED1
missense mutants (e.g., p.Thr102Arg) are unable to bindneurofibromin and are unable to recruit neurofibromin to
the cell membrane.
Spred1 knockout mice show spatial learning deficits
in the Morris water maze and T-maze tests. In the cognitively more demanding stages of the T-maze, the heterozygous knockout mice performed at an intermediatelevel between those of the wild type and their homozygous knockout littermates.27 Both of these spatial learning tasks are hippocampus dependent. Knockout mice
showed a decreased synaptic plasticity in the hippocampus as reflected by a diminished long-term potentiation
after stimulation of the Schaffer collaterals.27 The exact
pathogenesis of the learning problems in Spred1knockout
mice is not known, but increased Ras-MAPK signaling inneurons is probably an important factor.27 These findings
are comparable with the learning and synaptic plasticity defects observed in Nf1+/− mice,28 thus stressing theimportance of the RAS-MAPK pathway in learning andmemory. The learning and synaptic plasticity defects inadult Nf1+/− mice can be acutely rescued with lovastatin
treatment.28 Similar experiments have not yet been reported in Spred1 knockout mice.
Medical Management and Surveillance
The phenotype associated with Legius syndrome ismilder than the NF1 phenotype, and less stringent medical follow-up seems to be appropriate for those with Legius syndrome. However, at the moment, clinical data are
available only from a limited number of individuals with
Legius syndrome, and certain rare complications might
have been missed. A correct diagnosis of Legius syndrome has important implications for prognosis, counseling, and prenatal diagnosis. The diagnosis of Legius syndrome may relieve the psychological burden in families
who would otherwise be expecting more serious NF1-

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related complications.9
Clinical cohorts of patients with multiple CALMs contain individuals negative for a pathogenic NF1 or SPRED1
mutation, suggesting that there probably remains a small
group of unclassified individuals with mutations in yet
unidentified genes. In sporadic cases, mosaicism for an
NF1 (or SPRED1?) mutation can be present, explaining
the presence of NF1 features in the absence of a detectable NF1 or SPRED1 mutation in white blood cells.17
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