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RESEARCH ARTICLE
X-Linked Intellectual Disability Related Genes
Disrupted by Balanced X-Autosome Translocations
Mariana Moys
es-Oliveira,
1
Roberta Santos Guilherme,
1,2
Vera Ayres Meloni,
1
Adriana Di Battista,
1
Claudia Berlim de Mello,
3
Silvia Bragagnolo,
1
Danilo Moretti-Ferreira,
4
Nadezda Kosyakova,
2
Thomas Liehr,
2
Gianna Maria Carvalheira,
1
and Maria Isabel Melaragno
1
*
1
Department of Morphology and Genetics, Genetics Division, Universidade Federal de S~
ao Paulo, S~
ao Paulo, Brazil
2
Jena University Hospital, Friedrich Schiller University, Institute of Human Genetics, Jena, Germany
3
Department of Psychobiology, Universidade Federal de S~
ao Paulo, S~
ao Paulo, Brazil
4
Departament of Genetics, Instituto de Biocincias de Botucatu, Universidade Estadual de S~
ao Paulo, S~
ao Paulo, Brazil
Manuscript Received: 30 April 2015; Manuscript Accepted: 10 July 2015
Detailed molecular characterization of chromosomal rearrange-
ments involving X-chromosome has been a key strategy in
identifying X-linked intellectual disability-causing genes. We
fine-mapped the breakpoints in four women with balanced X-
autosome translocations and variable phenotypes, in order to
investigate the corresponding genetic contribution to intellec-
tual disability. We addressed the impact of the gene interrup-
tions in transcription and discussed the consequences of
their functional impairment in neurodevelopment. Three
patients presented with cognitive impairment, reinforcing the
association between the disrupted genes (TSPAN7—MRX58,
KIAA2022—MRX98, and IL1RAPL1—MRX21/34) and intellec-
tual disability. While gene expression analysis showed absence
of TSPAN7 and KIAA2022 expression in the patients, the unex-
pected expression of IL1RAPL1 suggested a fusion transcript
ZNF611-IL1RAPL1 under the control of the ZNF611 promoter,
gene disrupted at the autosomal breakpoint. The X-chromo-
somal breakpoint definition in the fourth patient, a woman with
normal intellectual abilities, revealed disruption of the
ZDHHC15 gene (MRX91). The expression assays did not detect
ZDHHC15 gene expression in the patient, thus questioning its
involvement in intellectual disability. Revealing the disruption
of an X-linked intellectual disability-related gene in patients
with balanced X-autosome translocation is a useful tool for a
better characterization of critical genes in neurodevelopment.
Ó2015 Wiley Periodicals, Inc.
Key words: intellectual disability; X-chromosome; KIAA2022;
IL1RAPL1;TSPAN7;ZDHHC15
INTRODUCTION
Intellectual disability (ID) consists of a broad range of disorders
characterized by low general intellectual functioning (IQ below 70)
and reduced adaptive skills, which is diagnosed before the age of
18 years. ID can originate from environmental causes and/or
genetic anomalies, and its prevalence in Western countries is
estimated to be 1.5–2% [Leonard and Wen, 2002; Maulik et al.,
2011].
Although the human X-chromosome carries only about 4% of
the protein-coding genes in the human genome, X-chromosomal
defects are thought to account for 8–12% of ID in males [Ropers,
2010]. X-linked forms of ID (XLID) are easily identifiable because
of their characteristic inheritance pattern [Gecz et al., 2009]. This
has rendered the X-chromosome an attractive target for research
for the molecular causes of ID and, up to now, around 110 genes
were associated with XLID [Piton et al., 2013; Hu et al., 2015].
Several approaches have been developed for the identification of
genes responsible for XLID. The ascertainment of a patient with
Conflict of interest: The authors declare that they have no conflict of
interest.
Grant sponsor: Fundac¸~
ao de Amparo
a Pesquisa do Estado de S~
ao Paulo;
Grant number: FAPESP # 2011/51690-1.
Correspondence to:
Maria Isabel Melaragno, Department of Morphology and Genetics,
Universidade Federal de S~
ao Paulo, Rua Botucatu, 740 CEP 04023-900,
S~
ao Paulo, SP, Brazil.
E-mail: melaragno.morf@epm.br
Article first published online in Wiley Online Library
(wileyonlinelibrary.com): 00 Month 2015
DOI 10.1002/ajmg.b.32355
How to Cite this Article:
Moys
es-Oliveira M, Guilherme RS, Meloni
VA, Di Battista A, de Mello CB, Bragagnolo
S, Moretti-Ferreira D, Kosyakova N, Liehr
T, Carvalheira GM, Melaragno MI. 2015.
X-Linked Intellectual Disability Related
Genes Disrupted by Balanced X-Autosome
Translocations.
Am J Med Genet Part B 9999:1–9.
Ó2015 Wiley Periodicals, Inc. 1
Neuropsychiatric Genetics
both ID and a balanced chromosomal rearrangement affecting the
X-chromosome has proven to be particularly valuable in searching
for XLID genes [Zemni et al., 2000; Lossi et al., 2002; Kalscheuer
et al., 2003; Cantagrel et al., 2004; Najm et al., 2008]. In women with
balanced X-autosome translocations the normal X-chromosome is
preferentially inactivated; thus most X-linked genes remain func-
tional only in the derivative chromosomes. Therefore, a gene
disruption within the X-chromosomal breakpoint may result in
the absence of functional copies of the disrupted gene. In these
patients, the precise breakpoint recognition is fundamental for the
interpretation of the rearrangement’s clinical impact.
However, since balanced rearrangements do not result in gain or
loss of genomic material, they are undetected by chromosomal
microarray-based genome wide surveys [Talkowski et al., 2011].
Therefore, other methodologies are necessary for achieving break-
point mapping with a high resolution definition [Gribble et al.,
2007].
In the present study, the array painting method was applied to
determine the breakpoints in four women with balanced X-auto-
some translocations and variable phenotypes. The impact of the
gene interruptions in transcription was evaluated as well as the
consequences of the gene function impairment in neuronal devel-
opment and function.
SUBJECTS AND METHODS
Subjects
We studied four women with balanced X-autosome translocation
who originally presented to genetic or endocrinological public
services in S~
ao Paulo state, Brazil. Two patients (patient 2 and 4)
presented with primary amenorrhea and their endocrinological
and gynecological evaluation was presented elsewhere [Moyses-
Oliveira et al., 2015].
Eight female individuals with age ranging from 7 to 51 years old
were selected from healthy population as control individuals for
gene expression investigation. This study was approved by our
Institutional Ethics Research Committee (CEP 0394/10), and
performed after obtaining written informed consent from the
patients or their parents.
Neuropsychological Assessment
Diagnose of ID was based on standards IQ scales such as the
Wechsler Adult Intelligence Scale (WAIS-III) and the Wechsler
Intelligence Scale for Children (WISC-III), as well as on the
Vineland-II adaptive behavioral scale. Some patients were also
submitted to a battery of traditional neuropsychological tests
[Strauss, 2006], including measures of visual constructive skills
(Copy of the Complex Figure of Rey), verbal memory processes
(Rey Auditory Verbal Learning Test; Semantic Verbal Fluency),
attention functions (Continuous Performance Test) and executive
functioning (5-digits test; Phonological Verbal Fluency-FAS).
When the application of standard tests was not possible, due to
the severity of cognitive impairment, an interview with parents was
performed in order to understand functionality and family con-
ditions. When signs of autism were observed, the Childhood
Autism Rating Scale (CARS) was used.
Classical Banding Cytogenetic Analysis and
Clinical Report
Chromosome analyses with G-, and replication banding using
bromodesoxyuridine (BrdU), as well as NOR-staining, were per-
formed on 72-h lymphocyte cultures from the patients and, when
possible, from their parents according to standard procedures,
based on a total of 40 metaphase cells.
Patient 1 (Fig. 1a) was a 40-year-old woman, ninth child of a
non-consanguineous and healthy couple. Her parents were not
available for the study and no clinical data was available for her
early childhood, still it was known that her developmental mile-
stones were delayed. At 35 years, clinical evaluation showed short
stature (1.41 m; <3rd centile), normal weight for her size
(37.1 kg; BMI ¼19.5; 50th centile), microcephaly (head circum-
ference 49.5 cm; <3rd centile), and long face. Total skeleton
X-ray imaging revealed craniofacial disproportion, narrowed
intervertebral space between L4, L5, and S1, coxa vara, genu
valgo, metatarsus aductus and short distal phalanx of toes. The
cranial magnetic resonance imaging revealed cerebral atrophy.
The audiometry showed mild high frequency hearing loss with
ossicular chain disruption. WAIS-III results revealed an IQ at
borderline levels (IQ 77). According to scores obtained in the
Vineland-II scale, adaptive behavior concerning receptive lan-
guage, coping skills, and personal and domestic daily living skills
are more developed (1SD to 2SD) than expressive language or
socialization (interpersonal relationships) skills (<3SD). Gross
and fine motor skills, on the other hand, were at normal levels.
Although neuropsychological assessment showed overall deficits
in all measures (centile <1), analysis of the WAIS-III subtests
suggests potentialities related to visuospatial and motor skills
(Bock Design) and to differentiate between essential and nones-
sential details (picture Completion). She showed an apparently
balanced translocation between chromosomes X and 21 as fol-
lows: 46,X,t(X;21)(p11;p13).
Patient 2 (Fig. 1b) was a 26-year-old woman, second child of a
non-consanguineous and healthy couple. She was born at term
with a birth weight of 2,500 g (3–10th centile) and with
an undocumented length and birth head circumference. Her
developmental milestones were delayed. At 24 years the patient
presents with severe impairment in all cognitive and adaptive
behavioral domains, including total dependence for self-care
and communicative skills. Due to her severe condition, the
application of standard neuropsychological tests was not possible.
Furthermore, CARS results revealed the most severe level of
autistic behavior: she did not speak or communicate and was
unable to read or write. At the age of 25 years, the clinical
evaluation showed short stature (1.50 m; <3rd centile), micro-
cephaly (head circumference 52.5 cm; 3–10th centile), minor
facial dysmorphic features, such as attached ear lobes, progna-
thism and broad neck. Also the patient presents with primary
amenorrhea and hyperglycemia. The abdominal ultrasound im-
aging revealed mild hepatic steatosis. At 26 years, cranial magnetic
resonance imaging revealed brachycephaly and morphological
alterations of the temporal lobes. She showed a de novo apparently
balanced translocation between chromosomes X and 3 as follows:
46,X,t(X;3)(q13;q11)dn.
2 AMERICAN JOURNAL OF MEDICAL GENETICS PART B
Patient 3 (Fig. 1c) was a 42-year-old woman with normal
hallmark developmental milestones and primary amenorrhea.
Her parents were not available for the study. At the age of 41 years,
WAIS-III results revealed high average intellectual performance
(IQ 111). No relevant discrepancies were detected between verbal
(VIQ 112) and nonverbal (EIQ 110) domains. She showed an
apparently balanced translocation between chromosomes X and 9:
46,X,t(X;9)(q13;p11.1).
Patient 4 (Fig. 1d) was a 16-year-old girl, only child of a non-
consanguineous and healthy couple. She was born at term with
a birth weight of 3,800 g (75th centile and length of 50.5 cm
(50th centile). Her developmental milestones were delayed. At
eight years, congenital adrenal hyperplasia syndrome was
detected. At 14 years, she had a normal morphological clinical
evaluation. At the age of 16 years, intellectual assessment
showed a severe intellectual disability (IQ 48). The patient
had overall comprehensive and expressive language impair-
ments. At the time of her assessment, she was submitted
to daily life abilities training in an outpatient service for
people with ID. Additionally, she showed a de novo apparently
balanced translocation between chromosomes X and 19 as fol-
lows: 46,X,t(X;19)(p21.1;q13)dn.
FIG. 1. Patients’ facial features, partial G-banding karyotype and ideogram of the chromosomes involved in the rearrangements.
MOYS
ES-OLIVEIRA ET AL. 3
DNA Isolation and Genomic Array
Genomic DNA was extracted from peripheral blood leukocytes
using the Gentra Puregene kit (Qiagen-Sciences, Germantown,
MD). The Affymetrix Genome-Wide Human SNP Nsp/Sty 6.0
array (Affymetrix Inc., Santa Clara, CA) was used to detect copy
number variations, and the data were analyzed with the Genotyp-
ing Console 3.0.2 and Chromosome Analysis Suite (ChAS) soft-
ware (Affymetrix Inc.) based on GRCh37/hg19. To determine the
imbalance pathogenicity, gene content and overlapping benign
copy number variation (CNV) regions were considered, according
to the Database of Genomic Variants (DGV—http://projects.tcag.
ca/variation/).
Microdissection and Amplification of the
Derivative Chromosomes and Reverse
Chromosome Painting
Both derivative chromosomes involved in each patient’s translo-
cation were collected from lymphocyte culture derived metaphase
cells spread on coverslips as previously described [Liehr et al.,
2002]. Collection was done with extended glass micro-needles
controlled by a micro-manipulator under an inverted microscope.
Approximately ten copies of the entire derivative chromosome or
the chromosome arm affected by the translocations were collected.
The acquired DNA was amplified by degenerated oligonucleotide
primed polymerase chain reaction (DOP-PCR) [Backx et al.,
2007]. The chromosomal origin of the microdissected segments
was assessed by reverse chromosome painting (i.e., fluorescence
in situ hybridization - FISH - on normal chromosome spreads), as
previously described by Liehr et al. [2002].
Array-CGH of the Dissected and Amplified DNA
The dissected and DOP-PCR amplified derivative chromosomes’
DNA from the same patient—der(X) and der(A)—were labeled;
one of them with Cy3 and the other with Cy5, and hybridized to the
same CGH array SurePrint G3 (Agilent Technologies, Palo Alto,
CA) as previously described [Moyses-Oliveira et al., 2015]. Two
different slide designs were used: SurePrint G3 Custom CGH
860 k designed with 46,928 probes exclusive for the X-chromo-
some breakpoint definition, and SurePrint G3 Unrestricted 1 1M
standard manufacturer design from catalog (AMADID 021529)
that covers the entire genome, for the autosome breakpoint
definition.
FISH for Breakpoint Mapping Validation
Bacterial artificial chromosome (BAC) probes spanning the break-
point regions defined by the array-CGH results were selected
through the UCSC (University of California, Santa Cruz) genome
browser. The following probes were applied: RP11-714I4
(chrX:38,470,254–38,631,716), RP11-34P8 (chrX:73,914,389–
74,105,538), RP11-1072D12 (chr3:95,897,890–96,067,315), CTD-
3225H3 (chrX:29,125,517–29,260,404), RP11-790H16 (chr19:53,
164,395–53,352,424), and RP11-203K12 (chrX:74,592,669–
74,748,513). They were FITC (fluorescein isothiocyanate) or TRITC
(tetramethyl rhodamine isothiocyanate) labeled by nick-translation
and hybridized on metaphase spread [de Carvalho et al., 2008].
RNA Isolation and Real-Time RT-PCR
Gene expression analysis was performed by real time (RT)-PCR for
the coding sequences that were disrupted by the X-chromosomal
and autosomal breakpoints. Whole blood RNA was extracted from
the patients and control individuals using the PAXgene Blood RNA
MDx Kit (Qiagen-Sciences). RNA concentration and purity were
assessed using NanoDrop ND-1000 Spectrophotometer (Thermo
Fisher Scientific, Wilmington, DE) and integrity was assessed by
agarose gel electrophoresis. The cDNA was synthetized using
High Capacity cDNA Reverse Transcription (Life Technologies,
Carlsbad, CA) using 770 ng of total RNA. For quantitative RT-
PCR, Gene Expression Assays (Life Technologies) with TaqMan
probes were performed. The assays Hs00190284_m1 (TSPAN7
exons 1 and 2), Hs02339405_m1 (KIAA2022 exons 2 and 3),
Hs00907432_m1(ZDHHC15 exons 1 and 2), Hs00911606_m1
(ZDHHC15 exons 11 and 12), Hs00990788_m1 (IL1RAPL1 exons
8 and 9), and Hs01864525_s1 (ZNF611 exon 7) were used for the
study of the disrupted genes expression and the assays
Hs99999905_m1 (GAPDH) and Hs99999903_m1 (ACTB) were
used as internal controls. All samples of cDNA were assayed in
technical triplicate, in 12 ml reaction volumes with standard PCR
conditions of the 96-Well Fast Thermal Cycling (Life Technolo-
gies). The cycle threshold (CT) was determined during the geo-
metric phase of the PCR amplification plots, as recommended by
the manufacturer. Acquired data were analyzed using ViiA
TM
7
Software v1.2.2 (Life Technologies). First, a qualitative analysis was
performed to assess presence or absence of expression in patients
and controls. The presence of expression in controls was consid-
ered only when the TaqMan assay showed amplification in all
control individuals with a maximum CT value of 38 and maximum
CT standard deviation of 1.0. For the genes expressed in patients
and controls, relative differences in transcript levels were quantified
using the 2
–DDCt
method [Livak and Schmittgen, 2001] with the
patient sample as calibrator (patient’s 2
–DDCt
¼1). The gene was
considered downregulated in the patient when the controls’ 2
–DDCt
presented the minimum value equal or upper than 1.5 and the
standard deviation equal or lower than 0.5.
RESULTS
Banding cytogenetic analyses and genomic array indicated that all
patients presented balanced X-autosome translocations, without
cryptic genomic imbalances. In addition, as expected in balanced
X-autosome translocations, replication banding studies showed
that the normal X-chromosome was inactivated in all patients‘ cells
analyzed (data not shown).
Array-CGH of the microdissected and amplified DNA from the
derivative chromosomes was informative for all X-chromosomal
breakpoints and for two autosomal breakpoints. Those break-
points were mapped with a resolution range of 3.5–95.6 kb
(Table I) and five of them suggested disruption of gene sequences,
four in X-chromosome and one in chromosome 19. The array-
CGH was uninformative for the fine localization of breakpoints
4 AMERICAN JOURNAL OF MEDICAL GENETICS PART B
localized in heterochromatic regions, e.g., centromere and short
arm of acrocentric chromosomes.
The breakpoint definition for patient 1 is described in full in
order to illustrate the methods employed (Fig. 2), whereas results
for the others are summarized in Table I.
Patient 1
For patient 1, breakpoint definition by array painting revealed that
the X-chromosomal breakpoint in Xp11.4 may have disrupted the
3’UTR region of TSPAN7 gene (Table I, Fig. 2). The localization of
this breakpoint was validated by FISH, using RP11-714I4 probe,
which showed a split signal on each of the derivative chromosomes
(Fig. 2c). The TSPAN7 gene expression analysis with a probe for
exons 1 and 2 showed absence of expression in patient 1’s periph-
eral blood, while normal TSPAN7 expression levels were found in
all controls (CT mean ¼34.9; CT standard deviation ¼0.3). The
array painting method was uninformative for the autosomal break-
point since it was located in the heterochromatic short arm of
chromosome 21, a region not covered by array-CGH for technical
reasons. The NOR-staining showed a strong signal in the der(21)
(Fig. 2d), indicating that this break affected the distal portion of the
chromosome 21 short arm, in the 21p13 band.
Patient 2
For patient 2, breakpoint definition indicated that the X-chromo-
somal breakpoint in Xq13.3 disrupted the KIAA2022 gene at intron
1 and that the autosomal breakpoint in 3q11.2 did not affect any
gene coding sequences (Table I). The localization of both break-
points was validated by FISH, using RP11-34P8 (Xq13.3) and
RP11-1072D12 (3q11.2) probes. For each probe, a split signal
on each of the derivative chromosomes was observed. The gene
expression assay, using a probe for exons 2 and 3, did not detect any
KIAA2022 gene expression in patient 2’s peripheral blood, while
normal KIAA2022 expression levels were found in all controls
(CT mean ¼35.0; CT standard deviation ¼0.9).
Patient 3
For patient 3, array painting indicated that the X-chromosomal
breakpoint in Xq13.3 disrupted the ZDHHC15 gene at intron 10
(Table I); data was validated by FISH, which showed split signals in
both derivative chromosomes with RP11-203K12 (Xq13.3) probe.
The RT-PCR was performed with two probes, one for the region
upstream to the breakpoint (exons 1 and 2) and the other for the
region downstream to the breakpoint (exons 11 and 12). No
ZDHHC15 gene expression was detected in patient 3’s peripheral
blood, while all controls presented normal expression levels (exons
1 and 2 CT mean ¼37.8 and CT standard deviation ¼0.9; exons 11
and 12 CT mean ¼36.2 and standard deviation ¼0.2). The array
painting method was uninformative for the autosomal breakpoint
which was located in chromosome 9 centromeric region.
Patient 4
For patient 4, the array painting revealed gene disruptions
at both rearrangement breakpoints. The X-chromosomal
TABLE I. X-Chromosome and Autosome Breakpoints Definition by Array Painting Patient
Phenotype Karyotype Breakpoints’ genomic coordinates
Resolution of the breakpoint
definition
Gene disruption at the
breakpoint
1 Borderline intellectual disability,
microsomia and microceplaly
46,X,t(X;21)(p11.4;p13) chrX:38,547,528–38,551,017 3.5–kb TSPAN7
chr21—array painting not
informative
——
2 Severe intellectual disability,
autistic behavior and primary
amenorrhea
46,X,t(X;3)(q13.3;
q11.2)dn
chrX:73,977,371–74,073,029 95.6 kb KIAA2022
chr3:95,953,332–96,033,144 79.8 kb Intergenic breakpoint
3 Primary amenorrhea 46,X,t(X;9)(q13.3; p11.1) chrX:74,628,683–74,633,728 5.0 kb ZDHHC15
chr9—array painting not
informative
——
4 Severe intellectual disability and
congenital adrenal hyperplasia
46,X,t(X;19)(p21.1;
q13.4)dn
chrX:29,177,784–29,189,041 11.3 kb IL1RAPL1
chr19:53,228,520–53,242,689 14.2 kb ZNF611
MOYS
ES-OLIVEIRA ET AL. 5
breakpoint in Xp21.1 disrupted the IL1RAPL1 gene at intron 2
and the breakpoint in 19q13.41 may have disrupted the
ZNF611 gene affecting the region that comprises 5’UTR
element to intron 3. (Fig. 3a). The localization of both break-
points was validated by FISH, using CTD-3225H3 (Xp21.1)
and RP11-790H16 (19q13.41) probes. For each probe, a split
signal was observed on each of the derivative chromosomes.
The IL1RAPL1 gene expression analysis was performed with a
probe that accessed the region downstream to the breakpoint
(exons 8 and 9). The real time PCR detected IL1RAPL1 gene
expression only in the patient ́
s peripheral blood, but not in
controls.
For the ZNF611 gene expression study, the probe accessed
the transcript region downstream to the breakpoint (exon 7).
The RT- PCR analysis demonstrated an approximate 50% reduc-
tion in expression in the patient in comparison to normal individ-
uals (controls’ 2
–DDCt
mean ¼1.9; controls’ 2
–DDCt
standard
deviation ¼0.3; Fig. 3b).
DISCUSSION
Detailed molecular characterization of chromosomal rearrange-
ments involving X-chromosome have been a key strategy in
identifying XLID genes [Zemni et al., 2000; Lossi et al., 2002;
Kalscheuer et al., 2003; Cantagrel et al., 2004; Najm et al., 2008].
Here four new patients with balanced X-autosome translocation
and disruption of XLID-related genes are described. The clinical
outcomes of these rearrangements are heterogeneous, with differ-
ent levels of intellectual impairment, ranging from severe intellec-
tual disability and autistic behavior to normal cognition. For the
patients with intellectual disability, the gene disruption in the
X-chromosome is the most likely cause of their cognitive im-
pairment, however other causative genetic variants cannot be ruled
out.
For the three disrupted XLID genes that are expressed in
peripheral blood under normal conditions (TSPAN7,KIAA2022,
and ZDHHC15), the RT-qPCR experiments showed absence of
FIG. 2. Breakpoint definition for patient 1. a: Ideogram of the chromosomes involved in patient 1’s rearrangement. b: Partial array CGH profile
obtained from the hybridization of the amplified microdissected derivative chromosomes. The X-chromosomal breakpoint is indicated by the
color change region, which results from the differential labelling of the two derivative chromosomes, causing high hybridization ratios in the
region distal to the breakpoint (probes in red in the upper graph and segment in blue in the lower graph) and low ratios in the region proximal
to the breakpoint (probes in green in the upper graph and segment in red in the lower graph). c: FISH in metaphase cells using RP11-714I4
probe, showing three hybridization signals, including split signals in the derivative chromosomes. d: NOR-staining showing a strong mark in
der(21).
6 AMERICAN JOURNAL OF MEDICAL GENETICS PART B
functional copies in the patients (patients 1, 2, and 3). Although it is
not possible to exclude the influence of environmental factors, the
gene expression investigation for these three genes suggested that
the rearrangements annulled their function in the patients‘ whole
blood.
Functional in vitro studies indicated that the TSPAN7 gene
(MIM #300210, MRX58), disrupted in patient 1, plays a role in
the maturation of glutamatergic synapses during neurodevelop-
ment [Bassani et al., 2012]. Loss-of-function of this gene has been
identified in males with mild to moderate intellectual disability
[Abidi et al., 2002; Maranduba et al., 2004]. Furthermore, this gene
has already been described as disrupted in another woman with
balanced X-autosome translocation associated to mild intellectual
disability [Zemni et al., 2000]. Although our patient presents an IQ
at borderline levels, this report reinforces the association between
TSPAN7 gene and neuronal development, since the neuropsycho-
logical assessment showed overall deficits.
Patient 2 does not have functional copies of the KIAA2022 gene
(MIM #300912, MRX98), which is highly expressed in the brain
and encodes a G-protein coupled purinergic receptor related to
neurite extension [Magome et al., 2013]. KIAA2022 loss of func-
tion, caused by chromosomal rearrangements or point mutations,
generally results in severe to profound intellectual disability and
autistic behavior [Cantagrel et al., 2004; Van Maldergem et al.,
2013]. Our patient presents with severe impairment in all cognitive
and adaptive behavioral domains associated to severe level of
autistic behavior, a similar phenotype to other patients previously
described with KIAA2022 functional impairment.
The ZDHHC15 gene (MIM #300577, MRX91), disrupted in
patient 3, encodes a palmitoyl acyltransferase, an enzyme that
promotes a post-translational modification critical for protein
localization and function in several cell signaling pathways [Young
et al., 2012]. The association between ZDHHC15 gene and XLID is
based on a previous report of one 29-year-old woman with a
balanced X-autosome translocation associated to severe intellec-
tual disability and ZDHHC15 disruption at exon 1, which caused
absence of gene expression in peripheral blood [Mansouri et al.,
2005]. However, the impact of ZDHHC15 gene in cognition has
never been replicated. Considering that the translocation in patient
3 disrupted and annulled ZDHHC15 gene expression in peripheral
blood but had no impact in cognition, the involvement of this gene
in intellectual disability is questionable. Although most studies for
the characterization of XLID-causing genes tend to focus only on
patients with cognitive impairment, patient 3 report showed that
the detailed genetic screening of subjects with normal cognition
and X-chromosome structural rearrangements might also be a
valuable resource to the better understanding of disease-causing
genes.
The molecular characterization of balanced chromosomal rear-
rangements involving the X-chromosome is definitely relevant in
searching candidate genes for diseases. However, the acclaim of a
new XLID-causing gene discovered by the study of a simplex case of
chromosomal rearrangement often lacks statistical power and can
lead to misleading conclusions [Piton et al., 2013]. Thus, the
validation of the involvement of ZDHHC15 gene in intellectual
impairment requires functional studies or replication of the same
genotype-phenotype associations in more patients.
The patient 4’s rearrangement affects the IL1RAPL1 gene (MIM
#300143, MRX21/34), which encodes a protein that is localized at
excitatory synapses and regulates exocytosis, the function of calci-
um channels and neurite elongation [Bahi et al., 2003; Gambino
et al., 2007; Valnegri et al., 2011]. The association between
IL1RAPL1 and cognitive function is well known. Mutations and
deletions affecting this gene have been frequently associated
to intellectual disability, autism and autism spectrum disorders
[Carrie et al., 1999; Sasaki et al., 2003; Wheway et al., 2003;
FIG. 3. Scheme of the patient 4’s rearrangement. a: Ideogram of the chromosomes involved in the rearrangement and genes disrupted at the
breakpoints (IL1RAPL1—NM_014271; ZNF611—NM_030972.3). b: Real-time PCR with 2–DDCt in patient 4 and controls, showing reduction of
ZNF611 gene expression in the patient. c: Scheme of a putative fusion transcript ZNF611-IL1RAPL1 under the control of the ZNF611 promoter
produced from the der(X).
MOYS
ES-OLIVEIRA ET AL. 7
Tabolacci et al., 2006; Bhat et al., 2008; Piton et al., 2008; Franek
et al., 2011; Youngs et al., 2012].
Although IL1RAPL1 gene is not expressed in peripheral blood in
normal conditions, transcripts from exons 8 and 9 of this gene
were detected in patient 4. Additionally, the gene disrupted at
the chromosome 19 breakpoint in patient 4, ZNF611, showed
decreased expression in the patient compared to controls. These
results suggest that a fusion transcript ZNF611-IL1RAPL1 under
the control of the ZNF611 promoter was produced from the der(X)
(Fig. 3c). The ZNF611 gene encodes a protein with a zinc finger
domain and unknown function, with is highly expressed in
peripheral blood [Hruz et al., 2008]. Although we cannot exclude
that ZNF611 haploinsufficiency played some role in patient 4’s
phenotype and even without the possibility of accessing the ner-
vous system, the patient’s intellectual impairment is probably due
to IL1RAPL1 loss of function.
CONCLUSIONS
This study emphasizes the precise breakpoints definition in bal-
anced X-autosome rearrangements as an important tool for search-
ing critical genes. The disruption of X-linked genes in patients with
cognitive impairment reinforces the involvement of the corre-
sponding genes in neurodevelopment. This strategy enabled the
confirmation of three XLID-causing genes (TSPAN7,KIAA2022,
and IL1RAPL1). Furthermore, the breakpoints fine mapping in
subjects with normal cognition might also lead to a better charac-
terization of candidate genes for diseases. This approach exposed
the necessity for validation of ZDHHC15 as an XLID-causing gene
and may encourage further studies to better characterize the
association between ZDHHC15 gene and intellectual disability.
For all XLID-genes assessed in this work, the description of
additional patients with similar genotype-phenotype correlations
would be useful for the better understanding of their role in the
neurodevelopment. Our findings provide new insights into the
XLID genetic landscape and might be useful for the molecular
diagnosis of intellectual disability in the future.
ACKNOWLEDGMENT
This study was supported by Fundac¸~
ao de Amparo
a Pesquisa do
Estado de S~
ao Paulo (FAPESP # 2011/51690-1).
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