Revealing the Complexity of a Monogenic Disease: Rett
Syndrome Exome Sequencing
Elisa Grillo1, Caterina Lo Rizzo1,2, Laura Bianciardi1, Veronica Bizzarri1,2, Margherita Baldassarri1,
Ottavia Spiga3, Simone Furini4, Claudio De Felice5, Cinzia Signorini6, Silvia Leoncini6,7,
Alessandra Pecorelli6,7, Lucia Ciccoli6, Maria Antonietta Mencarelli1,2, Joussef Hayek7, Ilaria Meloni1,
Francesca Ariani1, Francesca Mari1,2, Alessandra Renieri1,2*
1Medical Genetics, University of Siena, Siena, Italy, 2Genetica Medica, Azienda Ospedaliera Universitaria Senese, Siena, Italy, 3Biochemistry and Molecular Biology,
University of Siena, Siena, Italy, 4Department of Surgery and Bioengineering University of Siena, Siena, Italy, 5Neonatal Intensive Care Unit University Hospital Azienda
Ospedaliera Universitaria Senese (AOUS) of Siena, Siena, Italy, 6Department of Molecular and Developmental Medicine, University of Siena, Siena, Italy, 7Child
Neuropsychiatry Unit, University Hospital, AOUS, Siena, Italy
Rett syndrome (OMIM#312750) is a monogenic disorder that may manifest as a large variety of phenotypes ranging from
very severe to mild disease. Since there is a weak correlation between the mutation type in the Xq28 disease-gene MECP2/X-
inactivation status and phenotypic variability, we used this disease as a model to unveil the complex nature of a monogenic
disorder. Whole exome sequencing was used to analyze the functional portion of the genome of two pairs of sisters with
Rett syndrome. Although each pair of sisters had the same MECP2 (OMIM*300005) mutation and balanced X-inactivation,
one individual from each pair could not speak or walk, and had a profound intellectual deficit (classical Rett syndrome),
while the other individual could speak and walk, and had a moderate intellectual disability (Zappella variant). In addition to
the MECP2 mutation, each patient has a group of variants predicted to impair protein function. The classical Rett girls, but
not their milder affected sisters, have an enrichment of variants in genes related to oxidative stress, muscle impairment and
intellectual disability and/or autism. On the other hand, a subgroup of variants related to modulation of immune system,
exclusive to the Zappella Rett patients are driving toward a milder phenotype. We demonstrate that genome analysis has
the potential to identify genetic modifiers of Rett syndrome, providing insight into disease pathophysiology. Combinations
of mutations that affect speaking, walking and intellectual capabilities may represent targets for new therapeutic
approaches. Most importantly, we demonstrated that monogenic diseases may be more complex than previously thought.
Citation: Grillo E, Lo Rizzo C, Bianciardi L, Bizzarri V, Baldassarri M, et al. (2013) Revealing the Complexity of a Monogenic Disease: Rett Syndrome Exome
Sequencing. PLoS ONE 8(2): e56599. doi:10.1371/journal.pone.0056599
Editor: Osman El-Maarri, University of Bonn, Institut of experimental hematology and transfusion medicine, Germany
Received October 4, 2012; Accepted January 11, 2013; Published February 28, 2013
Copyright: ? 2013 Grillo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by ’Cell Lines and DNA Bank of Rett syndrome, X-linked mental retardation and other genetic diseases’ (Medical Genetics
Siena) - Telethon Genetic Biobank Network (Project No. GTB07001C to AR), by Ministero della Salute Grant No. RF-2010-2315597 - Ricerca Finalizzata 2010 and
Grant No. RF-TOS-2008-1225570 - Bando malattie rare to AR. This work was also supported by MIUR (PRIN 2008) to FM. The funders had no role in study design,
data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
The first publication of the catalogue of all known genes and
genetic disorders, Mendelian Inheritance in Man (MIM), in 1966,
fostered the idea that ‘‘rare diseases’’ were monogenic arising from
single or double mutational events in one of the 29,000 genes of
the human genome. On the contrary, ‘‘common diseases’’ are
thought to be complex deriving from interactions between
environmental factors and multiple mutational events in several
genes, as well as epigenetic modifications. Incomplete penetrance,
when individuals fail to express a trait, even when they have the
trait-allele, and expression variability, wherein traits are expressed
to different degrees among individuals with the same alleles, may
suggest that also supposedly monogenic diseases are more complex
than previously thought.
Rett syndrome (RTT) is a genetic neurodevelopmental
disorder that is characterized by regression especially in the
areas of language and motor abilities.  Studies have
implicated de novo mutations of the methl-CpG-binding protein
2 (MeCP2) gene on the X chromosome in RTT.  RTT has
a wide clinical spectrum.  Among the several hundred RTT
sporadic patients that we have studied we encountered two rare
familial cases consisting of pairs of sisters with RTT that are
phenotypically discordant. That is, individuals in each pair of
sisters demonstrate extremes of the RTT spectrum: classical
RTT and Zappella RTT variant (Z-RTT). .
One factor that can modulate X-linked disorders is X
chromosome inactivation (XCI) status.  However, all four
mentioned individuals have a balanced XCI, indicating that other
factors beyond XCI may contribute to the phenotypic outcome.
[3,5,6] Thus, these pairs of sisters represent the ideal model to test
the molecular basis of expression variability using an exome
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Materials and Methods
Two pairs of sisters with discordant phenotype were enrolled in
the study (Fig. 1a and 1b). Siblings #138 (classical RTT) and
#139 (Z-RTT) possessed the same mutation in MECP2,
c.1157del32, and showed a balanced XCI. The mutation was
inherited from their unaffected mother, who had a completely
skewed XCI.  Siblings #897 (classical RTT) and #896 (Z-
RTT) had an apparently de novo MECP2 deletion including exon 3
and part of exon 4.  XCI status analysis in this couple of sister
revealed balanced XCI in both.  The unrelated classical RTT
individuals #138 and #897 could not speak and walk and had
a profound intellectual deficit, while the Z-RTT individuals #139
and #896 could speak and walk and had a moderate intellectual
disability (Z-RTT). We quantified the striking differences in
somatic, neurodevelopmental, and neurovegetative features be-
tween the sisters using a previously described scoring system (score
from 0- mildest end to 40- most severe end; mean classical RTT
score of 27.565.3 and mean Z-RTT score of 13.865.9;
a threshold of 20 divided classical RTT from Z-RTT). 
According to this scoring system the classical RTT girls had
a clinical score of 30 (#138) and 33 (#897), which lies within the
range of scores for the most severe RTT outcomes. Conversely,
the Z-RTT girls had a score of 10 (#139) and 7 (#896) indicating
a milder, high functioning form of RTT (Table 1).  This study
was approved by the institutional review board of the University of
Siena (Siena, Italy). The parents of the patients have given written
informed consent, as outlined in the PLOS consent form, to
publication of their photograph. Participation in the study did not
alter the standard of care.
Exome Sequencing and Data Analysis
Whole exome sequencing (WES) was performed using the
Illumina platform in all 4 individuals (Methods S1 in File S1). Data
were filtered against dbSNP132 and control populations (1000
Genomes Project Consortium; http://www.1000genomes.org/
data). A further filtering was performedto retrieve only variants
potentially altering protein function, according to predictive tools,
i.e. truncating, splice site variants, and missense mutations
probably alter protein function (Methods S1 in File S1).
The clinical and genetic data of the two pairs of RTT sisters are
summarized in Table 1 and Fig. 1. Exome sequencing of 4 RTT
subjects, after filtering against dbSNP132 and control populations
(1000 Genomes Project Consortium; http://www.1000genomes.
org/data), revealed that in addition to the MECP2 mutation, each
patient had about 2500 variants, 330 of which exonic and splicing
changes. Using a combination of prediction tools, 82 variants per
patient were predicted to potentially impair protein function
(Tables S1–S3 in File S1). None of them were shared by the four
individuals. The variants were grouped on the basis of the
following criteria: i) exclusive to classical RTT girls (Table S1 in
File S1); ii) exclusive to Z-RTT girls (Table S2 in File S1); iii)
shared by two or three individuals with discordant phenotype
(Table S3 in File S1).
The first group includes 112 variants belonging to 108 genes
(Table S1 in File S1). Three genes, CNTNAP2 (OMIM*604569),
GFPT2 (OMIM*603865) and RYR1 (OMIM*180901) had varia-
tions predicted to impair the protein function in both the unrelated
classical RTT girls. These genes are involved in cell adhesion,
oxidative stress and calcium signaling. Each classical RTT patient
has in addition about 50 mutated genes among which we selected
21 potentially relevant genes through a meticulous analysis of the
literature on Pubmed and taking into account if the genes where
listed in OMIM and known to be associated with a neurological or
neuromuscular phenotype (10 genes) (Table 2) and if the related
protein was involved in a particular pathway (13 genes, 2 of which
were already selected using the above mentioned criteria) (Fig. 2a).
Interestingly, the two classical RTT patients shared alterations in
pathways of steroid biosynthesis, dopaminergic synapses, mRNA
surveillance and purine metabolism (Fig. 2a). Additional genes are
associated with muscle impairment and intellectual disability and/
or autism (Table 2).
The second group includes 80 variants/genes (Table S2 in File
S1), none of them shared by both the unrelated Z-RTT girls. On
the basis of shared pathway or disease association we selected an
additional 9 genes using the same criteria described for classical
RTT patients. Seven genes were selected on the basis of shared
pathway and, interestingly, a subset of these genes are related to
interleukine and chemokine receptors and, thus, may modulate
immune responses (Fig. 2b). Additional 5 genes were associated
with bipolar or metabolic disorders (Table 2). Three of them were
already selected on the basis of shared pathway.
The third group of genes includes 64 variants in 62 genes that
were shared by classical RTT and Z-RTT (Table S3 in File S1).
Among them, 46 were mutated in either one pair of discordant
sisters or the other.
Given the difference in the number of metabolic pathway genes
related to oxidative stress (OS) in classical versus Z-RTT patients,
we decided to test whether there was a difference in the OS
phenotype. Interestingly, for five out of six OS markers (non-
protein bound iron (NPBI), F(2)-dihomo-isoprostanes (F2-dihomo-
IsoPs), F(3)-isoprostanes, F(4)-neuroprostanes (F4-NeuroPs), and
F(2)-isoprostanes (F2-IsoPs)) there was not a statistically significant
difference between Z-RTT and controls, while in classical RTT
OS markers were significantly increased (Fig. 3).
RTT syndrome is usually due to de novo mutations in the MECP2
gene.  Therefore, the vast majority of cases are sporadic. The
two exceptional familial cases described here represent an ideal
model to identify genetic modifiers underlying expression
variability as in each couple there are two subjects manifesting
both ends of the phenotype (Table 1 and Fig. 1), and since each
couple will be enriched of identical variations facilitating the
selection of those not shared.
The most important finding of this study is that it demonstrates
that it is possible to use WES to gain insight into expression
variability in a monogenic disease such as RTT. We demonstrated
that each RTT subject had multiple mutations that may lead to
functional variants. Potentially, all the mutations have a role in
clinical manifestation and, despite our limited current knowledge
about the function of genes, we have defined a subset that may
cooperate to exacerbate (Table S1 in File S1) or ameliorate (Table
S2 in File S1) the final clinical outcome (Fig. 1).
Both patients with classical RTT had different heterozygous
missense mutations in the RYR1 gene, a regulator of Ca2+release,
which is responsible for a number of clinical conditions, including
a mild form of myopathy (Table S2 in File S1 and Fig. 2 and
Table 2). The RYR1 gene encodes the skeletal muscle ryanodine
receptor, which serves as a calcium release channel of the
sarcoplasmic reticulum, as well as being a bridging structure
connecting the sarcoplasmic reticulum and transverse tubule. 
RYR1 mutations have been associated with several congenital
Exome Sequencing of Rett Syndrome
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neuromuscular disorders. The RYR1 disrupting mutations identi-
fied in both classical RTT patients may contribute to the reduced
muscle mass, weakness, and susceptibility to scoliosis exibited by
classical RTT subjects but not in the Z-RTT patients.
Figure 1. Patient photographs and pedigree. In the pedigrees the two sisters couples are represented by grey circles (milder variant=Zappella
Rett variant (Z-RTT)) and black circles (more severe phenotype=classical Rett (RTT)). Panel a) Sisters #139 and #138 at the age of 28 and 19,
respectively, and pedigree. Presently, patient #139 is 40 years old and is still able to speak in short phrases. Although late stage RTT-associated motor
deterioration began 10 years ago, she is still ambulatory. Her phenotype was previously described. [3,35] Her sister, patient #138, is 29 years old and
has never been able to walk unassisted. Ten years ago she developed spastic tetraplegia with contractures that are still present and are further
deteriorating. Panel b) Sisters #896 and #897 at the age of 32 and 26, respectively, and pedigree. Presently, patient #896 is 39 year-old and is still
able to walk and to speak in short phrases. She has a friendly behavior and was extremely cooperative during examinations. Her somatic parameters
is in the mean range (Occipital-Frontal Circumference (OFC): 54.5 cm, 50–75thpercentile; height 162 cm, 25–50thpercentile; weight 63 Kg, Body Mass
Index (BMI)=24), she has a severe kyphosis and mild pes planus. She has no hand stereotypes and possesses good manual abilities, being able to
make simple drawings, eat independently, dress and wash herself. She has never had epilepsy, gastroesophageal reflux, breathing disorders and cold
extremities. She has bruxism and a high pain threshold. Her 34 year-old sister (patient #897) shows spastic tetraplegia with severe contractures and
hyperventilation. She shows somatic hypoevolutism (OFC 51,5 cm, ,3rd percentile; height 150 cm, ,3rd percentile; weight 29 Kg, BMI=13),
lordosis, and mild pes planus. She has constant hand stereotypes (pill counting and hand-mouthing), sialorrhea, bruxism, epilepsy that was not
controlled by therapy, and cold extremities. She has never been able to speak.
Exome Sequencing of Rett Syndrome
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Abrahams et al. noted that human CNTNAP2 expression was
enriched in circuits involved in higher cortical functions, including
language.  CNTNAP2 has been identified as an autism-
susceptibility gene and recessive mutations cause Pitt-Hopkins-
like syndrome 1 (OMIM#610042) and Cortical dysplasia-focal
epilepsy syndrome (OMIM#610042 ). A Cntnap2 knockout mouse
model revealed neuronal migration abnormalities, a reduced
number of interneurons, and abnormal neuronal network activity.
 These mice also demonstrated deficits in the three core
behavioral domains for Autism Spectrum Disorders (ASD), as well
as hyperactivity and epileptic seizure. Treatment with the FDA-
approved drug risperidone ameliorates the targeted repetitive
behaviors in the mutant mice. These data demonstrated a func-
tional role for CNTNAP2 in brain development and provide a new
tool for mechanistic and therapeutic research in ASDs.  In our
study CNTNAP2 mutations were observed in both classical
subjects, but not in their Z-RTT sisters (Table S2 in File S1 and
Fig. 2). Therefore, risperidone treatment may be a potentially
strategy for the treatment of classical RTT patients.
We have previously reported a duplication in the 1q42.12
regionin the Z-RTTpatient
(OMIM*609061).  This gene product localizes to cell-substrate
adhesion sites and sites of dynamic actin assembly and disassembly
participating in axonal outgrowth, dendrite morphology, synapse
formation, and axon guidance. Although ENAH mutations are not
listed in Tables S1–S3 in File S1, classical RTT patient #138 had
a 4bp insertion (insAAAC) in the UTR3 region of the ENAH gene
(position 225,675,743), at a site that is predicted to be conserved
(Phylo P =0.52) (the entire list of mutations is available on
request). This observation supports the role of ENAH in axon
guidance and in the modulation of the RTT phenotype.
Our results indicated that the classical RTT subjects are likely
to have a dysfunction in dopaminergic synapses due to functional
(OMIM*601647) genes encoding effectors of the postsynaptic
cascade that follow binding of dopamine to D5 (OMIM+126453)
receptor and D2 receptor (OMIM*126450), respectively (Table S1
in File S1 and Fig. 2a). This is in accordance with the finding that
there is a reduction in the number and soma size of tyrosine
hydroxylase-expressing neurons in a mouse model of RTT.  In
this model, L-Dopa treatment ameliorated the motor deficits. 
Interestingly, since dopamine D2-like partial agonists effectively
treat respiratory disorders in the same mouse model, ,
functional alteration of genes involved in dopaminergic synapse,
Table 1. Clinical features of the two couples of RTT sisters.
ITEM RTT sister pair 1RTT sister pair 2
Patient #138 Patient #139 Patient #896 Patient #897
Age of assessment 24 y33 y 39 y 8 m 34 y
Head (cm) 2-Microcephaly 0-No deceleration0-No deceleration 2-Microcephaly
Weight (kg)2-Below 3rdpercentile 0-Above 25thpercentile 0-Above 25thpercentile 2-Below 3rdpercentile
Height (cm)0-Above 25thpercentile2-Below 5thpercentile0-Above 25thpercentile2-Below 5thpercentile
Age of regression2-Before 18 months1-Before 18 months0-After 3 years0-After 3 years
Hand stereotypy 2-Dominating or constant1-Mild or intermittent0-None 2-Dominating or costant
Voluntary hand use2-None 0-Quite good hand use 0-Quite good hand use2-None
Sitting 0-Sitting unsupported at
age of 5
0-Sitting unsupported at
age of 5
0-Sitting unsupported at
age of 5
1-Loss of ability to sit
Walking2-Never learned to walk 0-Walking unsupported at
age of 5
0-Walking unsupported at
age of 5
1-Loss of ability to walk
Age of walk 0-Before 18 months0-Before 18 months1-After and equal to
1-After and equal to 18 months
Speech2-Never spoken0-More than 10 words at
age of 5
0-More than 10 words at
age of 5
Age of increasing words2-Never 0-Before 6 years1-After 6 years 2-Never
Level of speech 2-Absent 0-Phrases 0-Phrases2-Absent
Level of phrases2-Absent 1-Simple phrases1-Simple phrases2-Absent
Epilepsy1-Controlled by therapy0-No epilepsy at age of 50-No epilepsy at age of 52-Barely or not controlled
Breathing disorders 2-Severe0-Absent 0-Absent 2-Severe
Cold extremities1-Mild 1-Mild0-Absent 2-Severe
Sphincter control 1-Partial 0-Complete0-Complete 2-Absent
Genu valgu/Pes planus1-Mild1-Mild 1-Mild2-Severe
Kyphosis0-Absent 1-Partial 2-Severe 0-Absent
Scoliosis0-Absent 1-Mild0-Absent 0-Absent
Intellectual disability 2-Non measurable: IQ,20 1-Severe IQ: 20–401-Severe IQ: 20–402-Non measurable: IQ,20
TOTAL SCORE 30107 33
Exome Sequencing of Rett Syndrome
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ATF6B and PPP2R5E, may exacerbate respiratory disorders
typically observed in the 2 classical subjects.  Hyperventilation
or breath holding was not noted in the two Z-RTT girls at any
examination. Our analysis suggests that classical RTT patients
may benefit from L-Dopa treatment more than their Z-RTT
Both classical RTT subjects also likely had a partial block in the
squalene catabolism, because of the presence of heterozygous
(OMIM*603414), which encode the proteins CP24A and
ERG24, respectively. CP24A metabolizes the step from the active
form of vitamin D, calcitriol, to the inactive derivative calcitriol.
Disruptive mutations in this enzyme may cause an increase in
levels of calcitriol, cholesterol, and squalene. ERG24 catalyzes the
step from 4-4dimethyl-cholesta-8,14,24-trienol to 14-demethyla-
nosterol and mutations in this enzyme may also cause squalene
accumulation. Very recently, it has been demonstrated that
a mutation in squalene synthesis was found by randomly mutating
a second genomic site in Mecp2-mutant mice, which was able to
increase life span and decrease other RTT-like symptoms
(Communication by Justice M, at 7th World Congress on Rett
Syndrome, New Orleans, 2012). The same authors demonstrated
that Mecp2 null mice develop non fatty acid liver storage disease
(NAFLD), which is likely due to the link between Mecp2 and
histone deacetylase-3 (Hdac-3, OMIM*605166) (Communication
by Ebert D, at 7th World Congress on Rett Syndrome, New
Orleans, 2012). Indeed, a recent report indicated that liver
deletion of Hdac-3 causes a metabolic syndrome and increases
enzymes involved in cholesterol and lipid synthesis. [14,15].
In the classical RTT subjects we observed mutations in the
TM7SF2 and CYP24A1 genes, the gene products of which are part
of a steroid cascade downstream from squalene epoxidase. Such
mutations may have resulted in a partial block in the squalene
metabolic pathway that, in concert with the MECP2 haploinsuffi-
ciency, may have contributed to squalene accumulation. Together,
these data support a possible role of modifier genes in cholesterol
biosynthesis in RTT, and open the possibility to treatment of the
patients with anti-cholesterolemic agents. Statins are a widely used
and approved drugs and using specific outcome measures one can
investigate whether this treatment may be effective in reducing
some of the clinical outcomes of classical RTT patients.
Evidence of enhanced OS and lipid peroxidation has been
reported in patients with RTT.[16-18] Furthermore, studies
performed on hippocampus of the murine RTT model, mentioned
above, showed increased oxidative burden, changes in mitochon-
drial function, and a more sensitive response to oxidative
challenge.  The molecular mechanisms linking the MECP2
gene mutation to the subsequent OS derangement are unknown to
date. Recently, partial rescue of some of the neurological defects in
RTT by v-3 polyunsaturated fatty acids (PUFAs) has been
reported.  In support of this, we identified in classical RTT
subjects variants predicted to impair protein function in several
genes involved in OS. GFPT2, which exhibited the same splice site
mutation in both classical RTT girls, exerts a protective effect
Figure 2. Relevant pathways of altered genes in classical Rett (a) and Zappella Rett variant girls (b). Only pathways in which at least two
altered genes were included, or where one gene was mutated in either both classical Rett (RTT) (a) or both Zappella Rett variant (Z-RTT) (b) patients
have been included. Genes that are involved in only one pathway are in white. Genes that are involved in more than one pathway are indicated with
the same color. For each pathway the code assigned in the Kyoto Encyclopedia of Genes and Genomes (KEGG) database is indicated (see File S1). For
each gene the mutation type is indicated.
Exome Sequencing of Rett Syndrome
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against H2O2toxicity in neuronal HT-22 cells.  AOX1(O-
MIM*602841), catalyzes the formation of superoxide and is
expressed in the ventral horn of the spinal cord, primarily in the
glial cells.  Lastly, we identified mutations in ASMT
(OMIM*300162), the gene product of which is involved in the
synthesis of melatonin, a potent antioxidant (Fig. 2). As well as the
genes reported in Fig. 2, we identified variations in other genes
related to OS. These included KCNJ14 (OMIM*603953),
a potassium channel whose expression is modified after oxidant
exposure; RICTOR (OMIM*609022), a component of mTOR
complex 2 whose expression is regulated by Sirtuin1, whose
deficiency caused hepatic glucose overproduction, chronic hyper-
glycemia, and increased reactive oxygen species (ROS) pro-
duction; and ATF6B, involved in the unfolded Protein Response
pathway; and RYR1 itself, as in RYR1related miopathies oxidant
activity, the presence of OS markers and excessive production of
oxidant by mithocondria has been shown. [23–25].
Using NPBI, 4HNE-PAs, and several isoprostanes (IsoPs)
families as markers of redox derangement and lipid peroxidation,
we confirmed our previous data demonstrating that OS is present
in the classical RTT patients, while Z-RTT cases are more similar
to controls (Fig.3). [26,27].
The major novelty of the oxidative findings reported in the
present study is that RTT patients with identical MECP2
mutation, as our two pairs of sisters, can exhibit a different
pattern of OS markers according to their clinical phenotype (i.e.,
concordant genotype with discordant phenotype). While confirm-
ing the co-existence of a significantly increased pro-oxidant status
in genetically unrelated classical RTT subjects, the present data
suggest that the redox alteration observed in RTT is likely to be
modulated by genetic modifier factors, yet to be clarified. Earliest
markers of hypoxia (NPBI), as well as those markers indicating
general (F2-IsoPs) brain oxidative damage and specific grey (F4-
NeuroPs) and white (F2-dihomo-IsoPs) matter injury, were
elevated in classical RTT (Fig. 3). Interestingly, brain white
matter damage has been previously reported in RTT; this supports
the involvement of astrocytes in RTT, and their potential as
therapeutic targets. [28,29].
Table 2. Variations predicted to impair protein function in disease/susceptibility genes related to muscle and brain.
Gene Mutation type
(patient #) Trait-related molecular mechanism Susceptibility to
ABCA13 missense Heterozygous (897)Unknown Schizophrenia, bipolar
AP4M1splicing Heterozygous (138)Neuroaxonal damage and glutamate
/ Spastic paraplegia and severe
mental retardation (AR)
ATRN missense Heterozygous (138) Causes obesity by mimicking
CNKSR2 missense Heterozygous (897) Unknown/ Non-Syndromic Intellectual
CNTNAP2 missense Heterozygous
/Autism susceptibility 15 Pitt-Hopkins like syndrome 1 (AR),
Cortical dysplasia-focal epilepsy
DIRAS2 missense Heterozygous (897) Unknown Attention deficit/
Heterozygous (897)Unknown Autism/
KIF7missense Heterozygous (138)Regulation of GLI transcription factors
in SHH signaling pathway
/ Acrocallosal syndrome (AR)
Hydrolethalus syndrome 2 (AR)
Joubert syndrome 12 (AR)
RYR1 missense Heterozygous
Calcium signaling determining
contraction of skeletal muscle
Malignant hyperthermiaCentral core disease (AD and AR),
Minicore myopathy with external
congenital, with uniform type 1
TTC3missense Heterozygous (138) Inhibition of neuronal differentiationDown syndrome/
ANK3missense Heterozygous (139)Synapse formationBipolar disorder/
ASL missense Heterozygous (896)Detoxification of ammonia via the
/ Argininosuccinic aciduria (AR)
COG7 missense Heterozygous (139)Intracellular transport and glycoprotein
/ Congenital disorder of
glycosylation, type II (AR)
CPOXmissense Heterozygous (139)Heme biosynthetic pathway/Coproporphyria (AD)
GLDC missense Heterozygous (139)Degradation of glycine which has
a neurotransmitter role
/ Glycine encephalopathy (AR)
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In the Zappella variant patients fewer affected genes are known
to be involved in the OS pathway i.e. ASL (OMIM*608310),
CPOX (OMIM*612732), and GLDC (OMIM*238300 ) (Fig. 2b).
This observation is consistent with the results obtained measuring
OS markers (Fig. 2). Z-RTT variants in fact behave as controls
except for the levels of plasma 4-HNE PAs. This is an indicator of
co-existing protein oxidation due to aldehyde binding in the
presence of lipid peroxidation and its increase would suggest
a milder chronic oxidative damage in Z-RTT possibly sparing the
central nervous system (CNS).
Taken together, our results suggest that the genetic background
underlying the MECP2 mutation is strongly associated with OS in
classical RTT patients and may contribute to a better un-
derstanding of the biological mechanisms for the observed benefits
of PUFA supplementation in classical RTT patients.  On the
opposite, our data seem to suggest that PUFA supplementation
would be less efficient for Z-RTT patients. Our unpublished
observations on a larger (n=13) Z-RTT popupation (J. Hayek,
unpublished data) appear to further support this speculation.
The role of the immune system in RTT has been demonstrated
by the fact that transplantation of wild-type bone marrow restores
wild-type microglia and arrests pathology in a mouse model of
RTT.  It is, therefore, very interestingly that disrupting
mutations in chemokines and chemokines receptors were found in
Z-RTT patients, but not in the classical RTT patients. Chemokine
receptor CCR10 (OMIM*600240) is known to be expressed in
astrocytes.  There is evidence suggesting that selected
chemokines can induce further chemokine synthesis in astrocytes,
providing a mechanism to amplify inflammatory responses in
CNS.  The IL28RA (OMIM*607404) gene has a frame-shift
mutation that probably prevents its binding to members of the
OMIM*605658), which belongs to the pro-inflammatory IL17
family of cytokines, likely leads to alterations in protein function.
We hypothesize that this combination of mutations along with the
MECP2 disruption may modulate the immune system in a clinically
favorable way. It is difficult to speculate on the exact role of this
modulation, since the mechanism by which bone marrow
transplantation exerts beneficial effects is unknown at present. Z-
RTT patients may potentially have a more pronounced in-
flammatory response. It is also possible that Z-RTT subjects have
a less efficient inflammatory response to internal or external
(adjuvant of vaccines) stimuli. A more active response to such
stimuli may worsen the CNS damage in classical RTT patients.
Treating classical RTT with immunomodulators, such as IL-10
Figure 3. Comparison of oxidative stress markers in classical Rett versus Zappella Rett variant. In classical Rett (RTT) patients (N=2), all
the examined oxidative stress (OS) markers were significantly increased compared to healthy controls (N=15, all females, mean age 36.564.2),
whereas Zappella Rett variant (Z-RTT) patients (N=2) behave as controls subjects except for plasma 4-HNE-PAs. Intra-erythrocyte and plasma non-
protein bound iron (NPBI) are markers of hypoxia with hemoglobin oxidation and subsequent heme iron release. Plasma 4-HNE PAs is a marker of
protein oxidation due to aldehyde binding from lipid peroxidation sources. F(2)-isoprostanes (F2-IsoPs) are the end-products of arachidonic acid
oxidation, a polyunsaturated fatty acid that is abundant in both brain grey and white matter. F(2)-dihomo-isoprostanes (F2-dihomo-IsoPs) derive from
oxidation of adrenic acid, a fatty acid abundant in white matter, specifically myelin. F(4)-neuroprostanes (F4-NeuroPs) are the end-products of
docosahexanoic acid, abundant in neuronal membranes. Statistical differences were evaluated using Mann-Whitney sum rank test, Kruskal-Wallis
analysis of variance (ANOVA) Two-tailed P-values are shown. Values are expressed as means 6 standard error means (SEM); intra-erythrocyte NPBI is
reported as nmol/ml erythrocytes suspension; plasma 4-HNE-PAs are expressed as arbitrary units (AU), while isoprostanes (IsoPs) are expressed as pg/
Exome Sequencing of Rett Syndrome
PLOS ONE | www.plosone.org7 February 2013 | Volume 8 | Issue 2 | e56599
(OMIM*124092) would be an innovative strategy worthy of
It has been reported that the type of MECP2 mutation and the
X-inactivation status influence the clinical outcome of RTT. 
However, in the current study each pair of sisters had the same
MECP2 mutation and XCI.  Thus, these two pairs of sisters
represent an ideal model to test additional factors that modulate
the expression variability.
It is well known that the genetic background of mouse models
can influence phenotypic expression. The mouse model developed
in 2001 successfully phenocopies a number of aspects of RTT,
whereas previous models have failed in this attempt. [32,33]
Presently, it would be interesting to compare the genetic back-
grounds of mice (employed in previous mouse RTT models) in
which MECP2 mutations do not produce the RTT phenotype with
that of the current model.  In doing so, the contribution of
alterations in the dopaminergic system or of the oxidative burden
and mitochondrial dysfunction may be confirmed. [11,19].
The study of familial cases of RTT offers the opportunity to
identify the different molecular pathways involved in the
expression of discordant phenotypes. Our data show that
evaluating the degree of OS imbalance in patients with RTT
may also be important in fully understanding the disease
outcomes. OS status is known to be under the control of several
transcription factors and, in turn, plays a major role in cell
signaling and hence constitutes a potential phenotype modifier in
Together, our data indicate that the final phenotype in RTT
patients is likely the result of a combination of mutations in
MECP2, X inactivation status, and 40–50 disrupting variants in
other genes. Importantly, our study may have identified novel
targets for personalized RTT pharmacological intervention.
S1. Table S1, Variations predicted to impair protein function
exclusive to classical RTT patients. Table S2, Variations predicted
to impair protein function exclusive to Z-RTT patients. Table S3,
Variations predicted to impair protein function in discordant RTT
patients. References S1.
Supporting methods, tables, references. Methods
We would like to thank patients’ families for their participation in this study
and the Italian Rett Association (AIRETT). We thank Professors Thierry
Durand, Camille Oger, Jean-Marie Galano,
Alexandre Guy and Vale ´rie Bultel-Ponce ´ (Institut des Biomole ´cules Max
Mousseron (IBMM) - UMR 5247 CNRS, Montpellier, France) for
providing synthetized F2-dihomo-isoprostanes isomers. We also sincerely
thank professional singer Matteo Setti (www.matteosetti.com) for having
serendipitously triggered the scientific studies on redox alteration in Rett
girls. We thank Drs. Lesly Temesvari (Clemson University) and Karl
Franek (Bio-Scriptorium; www.bio-scriptorium.com) for their critical
reading of the manuscript and their editing improvements.
Conceived and designed the experiments: AR FM FA. Performed the
experiments: SL CS AP LC CDF. Analyzed the data: IM EG LB VB OS.
Contributed reagents/materials/analysis tools: SF CLR MB CDF MAM
JH. Wrote the paper: AR FM FA.
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Exome Sequencing of Rett Syndrome
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