Mental retardation and cardiovascular malformations in NF1 microdeleted patients point to candidate genes in 17q11.2.
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ABSTRACT: Neurofibromatosis Type 1 (NF1) is a frequent tumor suppressor gene disorder characterized by multiple benign tumors and high risk of malignancy. Internal tumor burden is a major disease-associated manifestation and can be most adequately assessed by magnetic resonance imaging of the whole body. Approximately 5% of NF1 patients have constitutional large NF1-deletions that are generally associated with more severe clinical manifestations. Here, we investigated whether these deletion patients also have more and/or larger internal tumors by assessing internal tumors and their total volume (exclusive of cutaneous and subcutaneous) in 38 NF1 deletion patients (including eight mosaic cases) and 114 age- and gender-matched NF1 patients without deletions. The incidence of internal tumors was significantly lower in mosaic deletion patients (1/8 = 13%) but did not differ between the 30 nonmosaic deletion patients and the 90 age- and gender-matched NF1 patients without large deletions used as controls. Neither the number nor the total volume of tumors per patient differed significantly between the latter two groups. However, extremely high tumor burden (>3,000 ml) was significantly more frequent among nonmosaic NF1 deletion patients than among NF1 patients without large deletions (13% vs. 1%, P = 0.014). Thus, as a group, patients with NF1 deletions do not exhibit a significantly higher internal tumor burden than NF1 patients without such deletions. However, deletion patients can frequently have extremely large internal tumors and thus demand special attention.Genes Chromosomes and Cancer 05/2012; 51(5):447-51. · 3.55 Impact Factor
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ABSTRACT: Patients with large deletions of the NF1 gene and its flanking regions (termed NF1 microdeletions) generally exhibit more severe clinical manifestations of neurofibromatosis type-1 (NF1). Here, we have investigated the clinical phenotype displayed by eight patients harbouring mosaic type-2 NF1 microdeletions. These patients did not exhibit facial dysmorphism, attention deficit hyperactivity disorder, delayed cognitive development and/or learning disabilities, cognitive impairment, congenital heart disease, hyperflexibility of joints, large hands and feet, muscular hypotonia or bone cysts. All these features have previously been reported to be disproportionately associated with germline (i.e. non-mosaic) type-1 NF1 microdeletions as compared with the general NF1 population. Plexiform neurofibromas were also less prevalent in patients with mosaic type-2 NF1 microdeletions as compared with patients carrying constitutional (germline) type-1 NF1 microdeletions. Five of the eight patients with mosaic type-2 deletions investigated here had 20-250 cutaneous neurofibromas, but only one of them exhibited a high load of cutaneous neurofibromas (N > 1,000). By contrast, a previous study indicated a high burden of cutaneous neurofibromas (N > 1,000) in 50% of adult patients with germline type-1 NF1 deletions. Patients with germline type-1 NF1 microdeletions have been reported to have an increased lifetime risk of 16-26% for a malignant peripheral nerve sheath tumour (MPNST). In this study, one of the eight investigated mosaic type-2 microdeletion patients developed an MPNST. We conclude that patients with mosaic type-2 NF1 microdeletions may also be at an increased risk of MPNSTs despite their generally milder disease manifestations as compared with germline type-1 NF1 microdeletions.Neurogenetics 05/2012; 13(3):229-36. · 3.58 Impact Factor
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ABSTRACT: Numerous disease syndromes are associated with regions of copy number variation (CNV) in the human genome, and in most cases the pathogenicity of the CNV is thought to be related to altered dosage of the genes contained within the affected segment. However, establishing the contribution of individual genes to the overall pathogenicity of CNV syndromes is difficult and often relies on identification of potential candidates through manual searches of the literature and on-line resources. We describe here the development of a computational framework to comprehensively search phenotypic information from model organisms and single-gene human hereditary disorders and thus speed the interpretation of the complex phenotypes of CNV disorders. There are currently more than 5000 human genes about which nothing is known phenotypically, but for which detailed phenotypic information for their mouse and/or zebrafish orthologs is available. Here we present an ontology-based approach to identify similarities between human disease manifestations and the mutational phenotypes in characterised model organism genes, and thus can be used even in cases where there is little or no information about the function of the human genes. We applied this algorithm to detect candidate genes for 27 recurrent CNV disorders and identified 802 gene/phenotype associations, approximately half of which were previously reported and half that were novel candidates. 431 associations were made solely on the basis of model organism phenotype data. Additionally, we observed a striking, statistically significant tendency for individual disease phenotypes to be associated with multiple genes located within a single CNV region, a phenomenon that we denote as pheno-clustering. Many of the clusters also display statistically significant protein functional similarity or vicinity within the protein-protein interaction network. Our results provide a basis for understanding previously un-interpretable genotype-phenotype correlations in pathogenic CNVs and for mobilizing the large amounts of model organism phenotype data to provide insights into human genetic disorders.Disease Models and Mechanisms 10/2012; · 4.96 Impact Factor
LETTER TO JMG
Mental retardation and cardiovascular malformations in
NF1 microdeleted patients point to candidate genes in
*M Venturin, *P Guarnieri, F Natacci, M Stabile, R Tenconi, M Clementi, C Hernandez,
P Thompson, M Upadhyaya, L Larizza, P Riva
J Med Genet 2004;41:35–41. doi: 10.1136/jmg.2003.014761
mutations of NF1, a tumour suppressor gene mapping to
17q11.2. Its main features include cafe ´ au lait spots, axillary
and inguinal freckling, iris Lisch nodules, neurofibromas, and
an increased risk of benign and malignant tumours,
particularly optic glioma, neurofibrosarcoma, malignant
peripheral nerve sheath tumours (MPNSTs),1and childhood
Over 70% of NF1 germline mutations cause truncation or
loss of the encoded protein.
Approximately 5–20% of all NF1 patients carry a hetero-
zygous deletion of usually 1.5 Mb involving the NF1 gene and
contiguous genes lying in its flanking regions,3 4which is
caused by unequal homologous recombination of NF1 repeats
(REPs).5Known as the ‘‘NF1 microdeletion syndrome,’’ this
condition is often characterised by a more severe phenotype
than is observed in the general NF1 group. In particular, NF1
microdeleted patients often show variable facial dysmorph-
isms, mental retardation, developmental delay, and an
excessive number of neurofibromas for age.3 6–12The severe
phenotype of microdeleted patients may be explained by
variations in the expression of the genes involved in the
rearrangement, which may be caused by different mechan-
isms, such as gene interruptions, position effects, and
decreased gene dosages.
different characteristics from those of classic NF1 patients,
it remains difficult to foresee the presence of the deletion
at an individual level on the basis of clinical observa-
tions. Various studies have reported the clinical char-
acterisation of NF1 deleted patients and the precise extent
of the deletion has been characterised in a subset.3–5, 13 14
However, no study comparing the incidence of specific
clinical signs in NF1 deleted and classical NF1 patients
has yet been published. The only published comparative
study concerned a single clinical sign (the development
of an MPNST), for which a correlation between NF1
microdeletion and a high risk for this tumour was
Our aims in the present study were, first, to verify whether
the incidence of specific clinical signs is different in NF1
microdeleted and general NF1 patients; and second, to
indicate possible correlations between the onset of distinct
clinical features and the haploinsufficiency of specific genes
involved in the deletions. We considered the extra-NF1
clinical signs shown by a sample of 92 microdeleted patients
(evaluated in this study or described in published reports),
and estimated their incidence in comparison with the NF1
patient group as a whole.
eurofibromatosis type 1 (NF1 [MIM 162200]) is a
common autosomal dominant disorder that affects
1/3500 individuals and is caused by deletion or point
In order to generate a database that was as comprehensive as
possible, we data-mined the NCBI Entrez Pubmed15and Med
Miner repository16and retrieved all the individually reported
cases of patients affected by the NF1 microdeletion syndrome
whose clinical phenotype was also described.
Signs included among the diagnostic criteria for NF1 were
excluded (with the exception of plexiform neurofibroma), as
were minor sporadically present signs for which no incidence
figures were available.
... ............ ............. ............ ............. ...........
Abbreviations: DSM-IV, Diagnostic and Statistical Manual of Mental
Diseases, fourth edition; FISH, fluorescent in situ hybridisation; MPNST,
malignant peripheral nerve sheath tumour; NF1, neurofibromatosis
N NF1 microdeletion syndrome is determined by haplo-
insufficiency of the NF1 gene and its flanking regions;
NF1 microdeleted patients show a more severe
phenotype than observed in classical NF1 patients.
N The aim of this study was to verify the presence of
specific clinical signs of NF1 microdeletion, by
combining clinical and genetic evidence from 92
deleted patients, 14 newly characterised and 78
N Statistical analysis, done by comparing the frequency
of 10 clinical signs between NF1 microdeleted patients
and the whole NF1 population, showed that the most
common extra-NF1 clinical signs in microdeleted
patients were learning disability, cardiovascular mal-
formations, and dysmorphisms.
N Using bioinformatic tools, the deletion gene content of
44 genetically and clinically characterised patients was
established. It is proposed that haploinsufficiency of
OMG and/or CDK5R1 genes may be implicated in
learning disability. In relation to cardiovascular mal-
formations, only JJAZ1 and CENTA2 can be consid-
ered as plausible candidate genes.
N When present in an NF1 patient, dysmorphisms,
cardiac anomalies, and learning disability are signs
indicating NF1 microdeletion.
This selection led to a total of 21 papers describing
individually reported cases for a total of 78 patients. We
excluded seven well characterised patients carrying mosaic
deletions from both published reports and the newly
The references of the extracted articles are 3–14 and 17–25.
Among the 78 patients described in published reports,
seven were familial microdeleted patients and in two cases
the parent showed a mosaic condition. The remaining
apparently sporadic patients can be considered founder
deletion carriers, although we cannot exclude low grade or
tissue specific mosaicism in the asymptomatic parents.
Conversely the 14 new NF1 deleted patients were recruited
by means of loss of heterozygosity (LOH) studies and
characterised by FISH (fluorescent in situ hybridisation)
analysis. Extension of FISH to the patients’ parents con-
tributing the deletion allowed us to identify a mosaic deletion
in parents of cases 65 and 94, and to exclude low grade
mosaicism in the remaining cases.
Both the newly described patients and those described in
the published reports fulfilled the NIH diagnostic criteria. We
classified microdeleted patients as being affected by mental
retardation only in those cases where intelligence quotient
(IQ) was reported or where an explicit statement of mild to
moderate to severe mental retardation was declared by the
investigators. When IQ was known, patients were classified
as having mild (IQ=50–70) or moderate to severe mental
retardation (IQ,50) according to the DSM-IV criteria.
With respect to cardiovascular malformations, we referred
to large surveys of NF1 patients investigated by conventional
methods for the diagnosis of cardiovascular malformations
(auscultation, radiography, electrocardiography, echocardio-
graphy), as these methods were applied to the NF1
microdeleted patients described.
The data on the percentages of each clinical sign in classic
NF1 patients were drawn from published reviews.2 26–29These
reference percentages may also include patients carrying the
NF1 deletion, the relative percentage of which is estimated to
The published reports and the recruited patients allowed us
to build a common data structure in which to tabulate the
information. For each patient, we added any new clinical sign
that had not been included previously, thus obtaining a
relational database with 103 fields.
The presence of a specific sign was attributed only when it
was explicitly reported and formalised in binary fashion (that
is, present or not present). When a field could not be
completed because of lack of information or an ambiguous
interpretation, it was defined as null and was not counted.
The analysed features were studied as discrete variables. As
the clinical data concerning each feature were not available
for all the patients, the total number of patients for whom the
data were applicable is given in each data entry. The
frequency of each sign was calculated as the ratio between
the evaluable patients and the affected patients, and the two
patient populations were statistically compared using the x2
test in 262 tables with one degree of freedom and a 0.1%
error probability (confidence range 99.9%).
Electronic database information
The proximal and distal boundaries of each kind of deletion
were defined, and the deletion specific gene content was
identified, using the integrated maps available on NCBI
(http://www.ncbi.nlm.nih.gov/genome/seq/) and UCSC (http://
Information concerning the expression patterns, the
presence of specific functional domains in the protein
products and their putative cellular role, and the existence
of hortologous genes in model organisms was obtained
from the following internet pages: LocusLink (http://
gene-encoded large proteins analyzed by Kazusa cDNA Project
(HUGE) (http://www.kazusa.or.jp/huge/), SAGE (http://www.
u-tokyo.ac.jp/), NCI60 cancer microarray project (http://
genome-www.stanford.edu/nci60/) and, for the homologous
The sequence homologies identified in Mus Musculus by
means of a BLAST search were confirmed using an analysis in
MGI and e! The Mouse Genome Sequencing Consortium Mouse
Genome Browser, in which the hortologous regions have been
mapped. The rat data were drawn from Rat genome data
The functional domain analysis for the proteins encoded by
the studied genes was undertaken using the tools and links
in the expert protein analysis system (EXPASY) molecular
biology server (http://www.expasy.ch/).
Clinical evaluation of NF1 patients
In order to verify the presence and incidence of specific
clinical signs in NF1 microdeleted patients in comparison
with those with classic neurofibromatosis 1, we considered a
sample of 92 microdeleted patients (14 novel clinical
descriptions and 78 from published reports).
Table 1 shows the clinical signs and symptoms on which it
was possible to make the comparative analysis. Among the
clinical signs found to be more frequent in NF1 microdeleted
patients than in the classic NF1 patients, there was a
significant difference (p,0.001, that is, 99.9%) in the
incidence of dysmorphic features, hypertelorism, mental
retardation, and cardiovascular malformations (table 1).
When available, we also extracted information on the
extent of the deletion when molecular cytogenetic character-
isation had been undertaken. Of the 92 microdeleted
patients, 44 underwent microsatellite or FISH and long range
polymerase chain reaction (PCR) analysis, including 28 for
whom the information was retrieved from published
reports,4 5 13 1414 described in the present study, plus two
previously reported cases that had been precisely charac-
terised by our group using FISH.3
Table 2 lists the clinical features of the 14 previously
unreported microdeleted patients, including those who
differed from the NF1 classical phenotype in the statistical
analysis. Four patients had short stature or retarded growth,
one had macrocephaly, and one was microcephalic. Only one
patient had excessive growth. Nine patients had dysmorph-
isms, only two had mild mental retardation, and three had
cardiovascular diseases. Examples of patients with facial
dysmorphisms from the newly described microdeleted group
are shown in fig 1.
The 44 finely characterised patients were then grouped on
the basis of the extent of their deletion to explore possible
genotype–phenotype correlations. Thirty seven patients car-
rying REP deletions made it possible to explore phenotypical
variability within a subset having the same deletion:
dysmorphic features, mental retardation, and cardiovascular
anomalies were present in, respectively, 34 of 37 patients
(92%), 12 of 26 (46.1%), and 7 of 37 (19%).
Eight patients with unusual sized deletions (one or both
endpoints not falling within the NF1 REPs) were a further
main resource for the genotype–phenotype correlation study
of NF1 patients carrying different deletions. They included
three patients (BL, 106-3, BUD)3 5 14 18carrying large deletions
that extended centromerically to REP-P and telomerically to
REP-M, all of whom suffered from mental retardation; two
(BL and 106-3) also had dysmorphic features, but only BL
had hypertelorism. Patients 113-1 and TOP5 14had small
deletions where the telomeric endpoint lies within REP-M
but the centromeric endpoint was localised 59 of the NF1
gene: both showed mental retardation and facial dysmorph-
isms (including hypertelorism in patient 113-1). Atypical
deletions included case 118 (present study)—who suffered
from seizures and in whom the telomeric boundary was
between NF1 intron 6 and exon 10b, whereas centromerically
in situ hybridisation (FISH) analysis
Clinical features of the 14 newly described patients carrying NF1 microdeletion characterised by refined fluorescent
malformation Other features
90th centile, macrocephaly
Height 3rd centile,
microcephaly 2nd centile
Short stature 10th centile
VSD (upper part)
Optic glioma, seizures
Broad neck, 3 NFs
Small hands/feet, short fingers
IQ54 Mitral insufficiencyMCLS, kyphoscoliosis, pectus
excavatum, genu valgus, pes
planus, umbilical hernia
Small hands/feet, short fingers
MCLS, amblyopia, thalamic
Hearing impairment, Noonan-like
NFs, required special education,
short and broad fingers and toes
Overgrowth .97th centile
–Delayed motor development,
short and broad feet, fifth finger
*Hypertelorism, epicanthic folds, low set ears, low posterior hairline.
?Hypertelorism, downslanting eye, strabismus, large and prominent nose with high and broad bridge, bulbous nasal tip, large and low set ears, malar hypoplasia,
wide and prominent philtrum, small mouth, small pointed chin.
`Hypertelorism, long philtrum, broad nose.
1Prominent forehead, hypertelorism, ptosis (O.DX), downslanting eyes; strabismus, large and prominent nose with high and broad bridge and bulbous nasal tip,
large and low set ears, wide and prominent philtrum, low posterior hairline.
?Coarse face, hypertelorism.
``Hypertelorism, broad and wild nasal bridge, broad nasal tip.
11Simple facial features.
??Epicanthic folds, bulbous nose, narrow high palate, low forehead.
cen-REP, deletion extending centromerically to REP-P; CVM, cardiovascular malformations; F, female; HCM, hypertrophic cardiomyopathy; LD, learning
disabilities; M, male; MCLS, multiple cafe `-au-lait spots; NF, neurofibroma; REP, NF1 repeat mediated deletion; U, unknown; VSD, ventricular septal defect.
according to published reports
Presence of specific clinical signs in 92 NF1 microdeleted patients v NF1 patients
NF1 microdeleted patientsNF1 patients
25 to 30
40 to 50
5 to 15
4 to 8
3.8 to 6
No (0.36 to 0.13)
No (1.6 to 6.48)
Yes (1065.8 to 264.6)
Yes (702 to 300.1)
No (7.11 to 1.5)
10 to 30
No (2.5 to 7.5)
*The discordant values between the two groups of patients and the relative clinical signs are given in bold.
?Including the following signs, each observed in at least one patient: coarse face, flat occiput/brachycephaly,
facial asymmetry, prominent forehead, frontal bossing, ptosis, downslanting deep set eyes, eversion of the lateral
eyelid, epicanthic folds, strabismus, large nose, prominent nose, high nasal bridge, broad nasal bridge, broad
nose, bulbous nasal tip, large ears, low set ears, malar hypoplasia, wide philtrum, prominent philtrum, small
mouth, thick lips, micrognathia, small pointed chin, low posterior hairline.
`Including: atrial septal defect, ventricular septal defect, patent ductus arteriosus, pulmonary stenosis, dilated aortic
valve, hypertrophic cardiomyopathy, mitral valve prolapse.
it extended beyond REP-P—and case 155-1,5whose deletion
ranged from the 59 of the NF1 gene to a breakpoint region
(also shared by BL and 106-3), and who had dysmorphic
features and mental retardation.
Deletion gene content analysis in NF1 patients
On the basis of the deletion characterisation of 44 patients
(16 analysed in our laboratory and 28 described by other
investigators), we identified a critical genomic interval
(fig 2)5 30; the only exception was patient BUD, who had a
deletion extending beyond the most telomeric ACCN1 gene
The genomic interval comprises 21 genes with a known
function, 10 with an unknown function, and 30 with
predicted functions supported by mRNA or EST alignments
with the genomic contig. The genes with a known function
are shown in fig 2.
As dysmorphisms, mental retardation, and cardiovas-
cular malformations were found to be commonly present
in the NF1 microdeleted subgroup in comparison with
the NF1 non-deleted patients, we searched the deleted
region for candidate genes that might be involved in
producing clinical signs such as mental retardation and
cardiovascular malformations, defined on the basis of the
target tissue or organ—that is, the central nervous system
and the heart. By combining database screening and
published findings concerning gene expression patterns
insufficiency may be involved in the onset of mental
retardation (SLC6A4, OMG, RHBDL4, ZNF207, CDK5R1, and
ACCN1), and two possible candidates for cardiovascular
malformations (CENTA2 and JJAZ1). In particular, the
oligodendrocyte-myelin glycoprotein (OMG) gene, which
maps within the REP interval (fig 2), encodes for a protein
that has been recently shown to be a potent inhibitor of
The solute carrier family 6 (serotonin neurotransmitter
transporter) member 4 gene (SLC6A4) (fig 2) maps centro-
merically to REP-P; its product is a transporter involved in
the uptake of the serotonin neurotransmitter by presynaptic
neurones or glial cells.32
The remaining candidate genes for mental retardation are
shared by the non-REP deletions extending telomerically to
REP-M (fig 2).
A good candidate for mental retardation is the cyclin
dependent kinase 5 regulatory subunit 1 gene (CDK5R1),
which encodes a neurone specific activator of cyclin
See table 2 for details of the facial dysmorphisms.
Facial appearance of three patients with NF1 microdeletions: case 116 at age 6 years (A), case 65 at age 6 (B), and case 72 at age 7 (C).
genes with a known function are shown in the upper line: the candidate genes for mental retardation and cardiovascular malformations are respectively
boxed and underlined; the black boxes represent NF1 REP-P and REP-M. The white, grey, and black circles at each deletion interval indicate absent,
mild, and moderate to severe mental retardation, respectively. The white and black squares indicate the absence and presence of cardiovascular
malformations. The frequencies of the conditions related to the above clinical signs are also given for the group of patients (n=37) carrying an REP
deletion. For the unusually sized deletions, the specific patient codes are indicated on the left.
Mapping to 17q11.2 region, from SLC6A4 gene to ACCN1 gene, of REP and unusual deletions from 44 NF1 microdeleted patients. All the
dependent kinase 5 (CDK5) required for the proper develop-
ment and functioning of the central nervous system.33 34In
addition, the neuronal amiloride sensitive cation channel 1
(ACCN1), zinc finger protein 207 (ZNF207), and rhomboid
veinlet-like 4 (RHBDL4) genes—which respectively encode a
neurone specific member of the degenerin/epithelial sodium
channel (DEG/ENaC) superfamily, a zinc finger protein, and a
protein homologous to the D melanogaster transmembrane
Rhomboid protein35–38—are all strongly expressed in the
central nervous system.
The Joined to JAZF1 (JJAZ1) and centaurin-a 2 (CENTA2)
genes, which are significantly expressed in the heart and
candidates for cardiovascular anomalies, were found to be
included in the REP deletion interval (fig 2).
In this study we considered the clinical signs not included
among the NIH consensus diagnostic criteria in a sample of
92 microdeleted patients, and compared their incidence with
that given for classical NF1 patients. We also established the
gene content of 44 deletions of known extent, and sought to
identify distinct clinical sign–genotype correlations.
Over the last few years, several papers have reported a more
severe phenotype in patients carrying a microdeletion than
in those affected by mutational neurofibromatosis,1 3 5 8–12
although, as pointed out by Tonsgard et al,10phenotype
evaluation per se is not predictive of the microdeletion.
By comparing a large sample of NF1 microdeleted patients
with the published data on classical NF1 patients, we
attempted to define the differences in the incidence of the
selected clinical signs between the two populations. When
selecting the clinical characteristics, we excluded all the signs
and symptoms that are diagnostic criteria for NF1, in order to
identify those that might highlight the candidate genes in
NF1 microdeletion syndrome. One exception to this rule was
plexiform neurofibroma, for which we considered the latest
emerging correlations between microdeletions and the
development of malignancy in the tumour.1Conversely,
although a high incidence of neurofibromas has been
reported in microdeleted patients, we did not include the
age dependent sign of neurofibroma development because of
the heterogeneity of the sample and the frequent lack of
information about neurofibroma onset.
We were aware that we may have underestimated the
difference in the incidence of the selected clinical signs
between classic NF1 and NF1 deleted patients because the
more recently identified and characterised patients with
deletions are included in the general NF1 population
evaluated in previous published reports.
The results of our study suggest a significantly higher
frequency of dysmorphisms, hypertelorisms, mental retarda-
tion, and cardiac anomalies in microdeleted patients (table 1).
With regard to dysmorphisms, an ascertainment bias needs
to be considered because the patients sent for microdeletion
analysis are commonly affected by a visibly more severe
phenotype which includes dysmorphic traits, whereas these
may be present but not reported in non-deleted NF1 patients.
This has also been shown recently in relation to other well
known microdeletion syndromes such as William’s and
Velocardio facial syndromes.39NF1 gene haploinsufficiency
is probably not the only cause of dysmorphisms, which are
likely to involve other genes in the complex pathways
regulating the correct development of the body as a whole.
It is currently impossible to correlate a single gene to such a
The only distinctive dysmorphic sign that was possible to
comparewith non-deletedpatients was hypertelorism
(table 1), although it may escape evaluation in the non-
deleted patients. It is easily detectable and therefore likely to
be reported more often than other signs. We agree with
Tonsgard on the difficulty of defining a specific dysmorphic
sign for NF1 microdeletion syndrome,10despite the consistent
general impression of a coarse and dysmorphic face. For all of
these reasons, we believe that no conclusions can be drawn
concerning the higher incidence of dysmorphisms in NF1
Another sign that was more represented in NF1 deleted
patients was mental retardation. It is worth noting that NF1
patients carrying large deletions have an increased frequency
of structural brain anomalies revealed by neuroimaging
studies, as shown by Korf and coworkers.40As these
anomalies are not usually seen in NF1 patients, it is
hypothesised that mental retardation may at least partially
reflect abnormal brain development rather than defective
brain function caused by neurofibromin haploinsufficiency.40
Zhu and coworkers41have shown that the cerebral cortex of
NF1-null mouse embryos develops abnormally, thus suggest-
ing the involvement of neurofibromin in CNS development.
NF1 patients rarely have a severe mental retardation (the
incidence is similar to that found in the general population,
at 3–5%), but often show a wide range of lesser mental
retardation and cognitive defects.42 43The significantly higher
incidence of moderate to severe mental retardation in
microdeleted patients probably reflects the haploinsufficiency
of one or more contiguous genes in addition to NF1.
We identified six candidate genes for mental retardation in
the deletion intervals, of which OMG and CDK5R1 are
particularly interesting because of their known function in
CNS development. CDK5R1 encodes a neurone specific
activator of cyclin dependent kinase 5.44Cdk5r KO mice have
severe cortical lamination defects and suffer from adult
mortality and seizures.33 34Moreover, an active CDK5-p35
complex is present in Golgi membranes, and antisense
oligonucleotide suppression of Cdk5 or p35 blocks the
formation of membrane vesicles from the Golgi apparatus
in young cultured neurones.45These results suggest that
Cdk5-p35 plays a role in membrane trafficking during the
outgrowth of neuronal processes.
It has recently been shown that OMG is a potent inhibitor
of neurite outgrowth that acts by binding to the Nogo
receptor, a protein associated with myelin.31Interestingly,
OMG lies within an NF1 intron, and the fact that its
expression pattern overlaps that of NF1 suggests that the
activity of the two genes might be under coordinated
control.46The deletion of the entire NF1 gene (and therefore
OMG) may deregulate this control mechanism and thus
contribute to the mental retardation outcome in microdeleted
We also compared the presence and severity of mental
retardation with the different deletion intervals with pre-
cisely mapped end points. As summarised in fig 2, 38.6% of
the patients carrying an REP deletion have mental retarda-
tion, but only 7.7% have moderate to severe mental
retardation. On the other hand, all of the four patients with
a deletion extending telomerically to the REP-M are affected
by moderate or severe mental retardation, which may
indicate that haploinsufficiency of one or more genes distally
to REP-M, such as CDK5R1, plays a critical role in brain
function or development, thus accounting for the onset of
mental retardation in patients carrying such deletions. The
hypothesis that severe mental retardation is indicative of a
deletion extending telomerically to REP-M needs to be
confirmed by parallel clinical and genetic characterisations
of a larger number of microdeleted patients.
In relation to cardiovascular involvement, several papers have
recently highlighted the presence of cardiac and cardiovas-
cular anomalies in patients with neurofibromatosis; in
particular, Friedman et al have underlined the recurring
cardiovascular anomalies that should be investigated in all
patients with a diagnosis of NF1.47It has also been reported
that neurofibromin plays a role in heart development,48and
that NF1 mutations should be taken into account as a cause
of cardiac malformations. Our sample indicates a much
higher incidence of cardiac malformations in microdeleted
patients, thus suggesting a possible contribution to correct
cardiac development of at least one of the other deleted genes
contiguous to NF1.
All the 11 patients with cardiovascular malformations
carry a REP deletion, thus indicating the possible presence
within this region of one or more genes involved in the
development of the cardiovascular system. Currently, the
available functional data concerning the genes included in
REP intervals do not allow us to identify genes that are
possibly involved in heart development. We did, however,
consider CENTA2, which encodes a phosphatydilinositide
binding protein,49and JJAZ1, a zinc finger containing
protein,50as candidates because of their high level of
expression in heart tissue.
Further in silico and expression studies are in progress to
identify genes with a known or unknown function that map
in the interval of typical and atypical deletions and may be
involved in heart development.
Dysmorphisms, cardiac anomalies, and mental retardation
are signs which, when present in an NF1 patient, should lead
to the suspicion of a microdeletion involving the NF1 and
contiguous genes. On the basis of our data, the more severe
phenotype is probably caused by the loss of other contiguous
genes as well as by NF1 haploinsufficiency.
It should also be considered that, in addition to the
deletion itself, the variation in the level of expression of the
genes involved in the rearrangements may also be caused by
additional mechanisms, such as gene interruption and the
position effect of genes flanking the deletions. Modulation of
the overall clinical phenotype associated with specific
polymorphisms has been described in Velo cardiofacial
syndrome,51and additional genetic factors are probably
involved in the clinical phenotypic variations observed in
patients carrying a similar REP deletion.
As the number of the microdeleted patients carrying REP
and non-REP deletions increases, more specific genotype–
phenotype correlations can be inferred and may validate the
differences we observed in the incidence of specific signs
between microdeleted and classic NF1 patients.
We thank Dr C Gervasini, Dr F Orzan, and P Colapietro for their
contribution to FISH analysis of the newly characterised NF1
microdeleted patients. This work was supported by a 2002 grant
from FIRST to PR and by a 2002 grant from AIRC to LL.
M Venturin, P Guarnieri, L Larizza, P Riva, Department of Biology and
Genetics, Medical Faculty, University of Milan, Italy
F Natacci, Medical Genetics Service, Istituti Clinici di Perfezionamento,
M Stabile, Medical Genetics Service, Cardarelli Hospital, Naples, Italy
R Tenconi, M Clementi, Clinical Genetics and Epidemiology Unit,
Department of Paediatrics, University of Padua, Italy
C Hernandez, Molecular Genetics Unit, Hospital Ramon y Cajal,
P Thompson, M Upadhyaya, Institute of Medical Genetics, University of
Wales College of Medicine, Cardiff, UK
*These authors contributed equally to the study
Correspondence to: Professor Paola Riva, Department of Biology and
Genetics, Medical Faculty, University of Milan, Italy; email@example.com
Received 22 September 2003
Accepted 2 November 2003
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Severe infantile hyperkalaemic periodic paralysis and paramyotonia congenita:
broadening the clinical spectrum associated with the T704M mutation in SCN4A
F Brancati, E M Valente, N P Davies, A Sarkozy, M G Sweeney, M LoMonaco, A Pizzuti, M G Hanna,
onset of episodes was unusually early, beginning in the first year of life and persisting into
adult life. The paralytic episodes were refractory to treatment. Patients described minimal
paramyotonia, mainly of the eyelids and hands. All affected family members carried the
threonine to methionine substitution at codon 704 (T704M) in exon 13 of the skeletal
muscle voltage gated sodium channel gene (SCN4A). The association between T704M and
the hyperPP/PMC phenotype has been only recently revealed. Nevertheless, such a severe
phenotype has never been reported so far in families with either hyperPP or hyperPP/PMC.
These data further broaden the clinical spectrum of T704M and support the evidence that
this mutation is a common cause of hyperPP/PMC.
m Journal of Neurology, Neurosurgery, and Psychiatry 2003;74:1339–1341.
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n Italian kindred is described with nine individuals affected by hyperkalaemic
periodic paralysis associated with paramyotonia congenita (hyperPP/PMC). Periodic
paralysis was particularly severe, with several episodes a day lasting for hours. The