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Non-syndromic language delay in a child with disruption in the Protocadherin11XY gene pair.

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RESEARCH ARTICLE
Neuropsychiatric Genetics
Non-Syndromic Language Delay in a Child With
Disruption in the Protocadherin11X/Y Gene Pair
Marsha D. Speevak,* and Sandra A. Farrell
Department of Genetics and Laboratory Medicine, Credit Valley Hospital, Mississauga, Ontario, Canada
Received 10 November 2010; Accepted 3 March 2011
Protocadherin11 is located on both the X and Y chromosomes in
Homo sapiens but only on the X chromosome in other hominid
species. The pairing of PCDH11Y with PCDH11X arose following
a duplicative 3.5 Mb translocation from the ancestral X chromosome to the Y chromosome several million years ago. The genes
are highly expressed in fetal brain and spinal cord. The evolutionary consequence of this duplication has been proposed to
include the sexual dimorphism of cerebral asymmetry and the
hominid specific transition to the capacity for language. We
report a case of a male child referred for genetic investigation of
severe language delay. Microarray analysis indicated the presence of a 220 Kb intragenic deletion at Xq21.31 involving the
PCDH11X gene. Fluorescence in situ hybridization using a BAC
probe mapping to intron 2 of the Protocadherin11X/Y gene pair
confirmed loss of the locus on both the X and Y chromosomes.
The X chromosome deletion was maternally inherited, but the Y
chromosome deletion was found to be a de novo occurrence in
this child. This finding lends support to the hypothesis that the
Protocadherin11X/Y gene plays a role in language development
in humans and that rare copy number variation is a possible
mechanism for communication disorders. 2011 Wiley-Liss, Inc.
Key words: oligonucleotide array; fluorescence in situ hybridization; FISH; PCDH11X/Y
INTRODUCTION
The Xq21.3/Yp11.2 block of homology between the sex chromosomes in humans has been traced back in human evolution to a
3.5 Mb duplicative translocation from the X long arm to the Y short
arm occurring approximately 6 million years ago and a more recent,
subsequent Y chromosome paracentric inversion [Schwartz et al.,
1998; Williams et al., 2006]. Included within this block are the
Protocadherin X and Protocadherin Y (PCDH11X/Y) genes, which
are members of the cadherin superfamily, both of which are highly
expressed in the fetal brain and spinal cord [Yoshida and Sugano,
1999; Blanco et al., 2000]. Confirmation that the X–Y homologous
status for PCDH11 seen in humans is absent in non-human
primates Pan troglodytes and Gorilla gorilla, supports the hypothesis
that these genes played a possibly significant role in the evolutionary
transition in modern humans to language and brain development
[Crow, 2002; Wilson et al., 2006]. X chromosome genes with
Y chromosome homology normally escape X inactivation. The
methylation status of PCDH11X has been studied in female humans
2011 Wiley-Liss, Inc.
How to Cite this Article:
Speevak MD, Farrell SA. 2011.
Non-Syndromic Language Delay in a Child
With Disruption in the Protocadherin11X/Y
Gene Pair.
Am J Med Genet Part B 156:484–489.
with results supporting escape from X-inactivation [Lopes et al.,
2006]; however replication timing studies were inconclusive
[Wilson et al., 2007].
We present here a case of a child with non-syndromic significant
speech delay, who was found by microarray and fluorescent in situ
hybridization (FISH) to have a small deletion within the PCDH11X/
Y gene pair. The X chromosome deletion was inherited from the
mother, but the Y chromosome deletion was a de novo occurrence.
This finding lends support to the hypothesis that this gene pairing
has an influential effect on the capacity for language in humans.
MATERIALS AND METHODS
DNA was extracted from an EDTA peripheral blood sample using a
Puregene DNA Extraction kit (Invitrogen, Carlsbad, CA). DNA
concentration was determined by Nanodrop (Thermo Scientific,
Wilmington, DE). Following enzymatic fragmentation, the DNA
was labeled with Cyanine5 and pooled male control DNA was
labeled with Cyanine3. The sample was hybridized to a 105 k array
(CytoChip 105, Bluegnome, Cambridge, UK) for 16 hr and washes
were performed using the Little Dipper (SciGene, Sunnyvale, CA).
The array was then scanned using a GenePix 4000B 5 mm scanner
(Molecular Devices, Sunnyvale, CA). The tiff image was analyzed
using the BlueFuse Multi software (Bluegnome).
The putative deletion was analyzed in silico using available
online databases. These included the UCSC Genome Browser
Human mar. 2006 (hg18) assembly (http://genome.ucsc.edu/
*Correspondence to:
Marsha D. Speevak, Genetics, Credit Valley Hospital, 2200 Eglinton Ave W,
Mississauga, ON, Canada L5M 2N1. E-mail: mspeevak@cvh.on.ca
Published online 7 April 2011 in Wiley Online Library
(wileyonlinelibrary.com).
DOI 10.1002/ajmg.b.31186
484
SPEEVAK AND FARRELL
485
index.html?org¼Human), the Database of Genomic Variants
(http://projects.tcag.ca/variation/) and the Decipher database
(http://decipher.sanger.ac.uk/). Structure was based on NM_032968.3 (PCDH11X, Gene ID 27328) and NM_032971.1
(PCDH11Y, Gene ID 83259) and was further analyzed using Alamut
(Interactive Biosoftware, Rouen, France).
A labeled BAC clone, RP11-339M12 (supplied by The Centre for
Applied Genomics, Toronto, Canada) was selected based on its
nucleotide position in relation to that of the deletion. An Xq
telomere probe (Abbott Laboratories, IL) was used to identify the
X chromosome. The Y chromosome was identified by the DAPI
bright long arm. Fluorescent in situ hybridization (FISH) was
performed on metaphase chromosomes prepared using standard
techniques.
CASE REPORT
The patient was seen in the genetics clinic due to speech delay. He
had a term, normal vaginal delivery. Birth weight was 2.805 kg, head
circumference was 33 cm and Apgar scores were 9 at 1 min and 9 at
5 min. The mother did not recall any neonatal complications. His
first words came at about age two and behaviorally, the child was
known to be aggressive. At age 3.5, his behavior included spitting
and rocking with excessive hand movements and solitary play.
At this time, a differential diagnosis of an autism spectrum
disorder was ruled out by psychiatric assessment, but no specific
diagnosis was suggested. He began to receive speech therapy at the
age of 4. During the genetic examination at age 4.5, he was able to
use only a few words in a sentence and they were not easily
understood. On examination, the height was about the 90th
percentile and the weight approximately the 50th percentile. The
head circumference was at the 10th percentile. A physical examination revealed no major dysmorphic or congenitally abnormal
features. Mild 2–3 toe syndactyly was noted, which apparently
his father and a paternal cousin were known to have. Fragile
X syndrome was ruled out by molecular studies. The other investigations were normal, including plasma amino acid screening,
urine amino acid screening, and urine for reducing substances.
The maternal family history was not contributory. To rule out
a chromosomal cause for the language delay, microarray studies
were ordered.
RESULTS
The microarray results indicated the presence of nine copy number
variants, varying in size from 37 to 218 Kb. Eight of these were well
represented in the Database of Genomic Variants, and interpreted
to be benign (Table I). The ninth variant was a unique deletion of 23
probes at Xq21. The size of the deletion was estimated at 218,375
bases. The closest proximal probe that was not deleted was 23,708
bases away, and the closest distal probe that was not deleted was
22,168 bases away. Thus, the maximum size of the deletion was less
than 264,251 bases. The deletion was described as arr Xq21.31
(91,035,601–91,253,976) 0 with outermost boundaries at
91,011,893 and 91,276,144. In silico analysis of the region revealed
the deletion was within intron 2 of the PCDH11X gene, which in its
entirety occupies base positions 90,976,315–91,764,882. The homologous gene on the Y chromosome, called PCDH11Y, is found at
band Yp11.2 (4,984,131–5,670,264), in the male specific region
(MSY) known as X-transposed. This region is 99% identical to a
3.5 Mb region of X chromosome at band q21, and contains only two
genes, PCDH11Y and TGIF2LY [Skaletsky et al., 2003]. The microarray findings are shown in Figure 1.
Fluorescence in situ hybridization (FISH) was performed on
cultured peripheral lymphocyte nuclei of the patient and his
parents, using BAC probe RP11-339M12, which maps to Xq21.31
at position 91,031,581–91,227,122 (intron 2 of PCDH11X)). The
hybridization patterns were consistent with a maternally inherited
X deletion and a de novo deletion within the short arm of the Y
chromosome, since the father had a normal hybridization pattern
(Fig. 2). The localization of RP11-339M12 to PCDH11Y intron 2
was confirmed by in silico comparison of the end and middle
sequences of RP11-339M12 with the sequence of PCDH11Y. RP11339M12 was found to be closely homologous to Y chromosome
bases 5,039,673–5,238,049 in intron 2 of PCDH11Y, approximating
the position of the probe to intron 2 of PCDH11X (Fig. 3). A nine
marker multiplex microasatellite marker comparison was performed (AmpF‘STR Profiler Plus, Applied Biosystems, Life Technologies, Carlsbad) on DNA samples from the patient, his mother
and his father in order to confirm paternity and thereby confirm
that the Y chromosome deletion was indeed de novo. The profiles
were consistent with the expected paternal inheritance at all loci
examined (data not shown). As well, based upon the array design,
TABLE I. Copy Number Variants Detected in the Patient, and Represented in the Database of Genomic Variants (DGV)
Region
1q44
3q29
4q13.2
6p21.32
14q21.2
14q32.33
22q11.21
22q11.21
Type
Loss
Loss
Loss
Loss
Loss
Loss
Gain
Gain
Left position
246806603
196886918
69057765
32558700
45533445
105708239
17041749
18782655
Right position
246852156
196924370
69165843
32660112
45693117
105873039
17119280
18989577
Size (Kb)
45.5
37.5
108.1
101.4
159.7
164.8
77.5
206.9
Within DGV variation
103996
91816
7448
111517
49366
10460
31070
79451
AMERICAN JOURNAL OF MEDICAL GENETICS PART B
486
FIG. 1. Homozygous deletion detected by oligo array analysis at Xq21.31:91,035,601–91,253,976 is shown with the corresponding Y chromosome
region.
the homozygous deletion could potentially include all of exon 2 of
PCDH11X/Y. However, PCR amplification of exon 2 of PCDH11X/
Y was consistent with retention of at least one copy of the exon and
its 30 boundary in the child and his parents (results not shown).
The final results of the experiments were described in FISH
terms as ish: del(X)(q21.31q21.31)(RP11-339M12-)mat, del(Y)(p11.2p11.2)(RP11-339M12-)dn and the case was interpreted in
the report as a variant of uncertain clinical significance.
DISCUSSION
Cadherins are a large family of genes responsible for cell–cell
interactions, including cell specific adhesion and cell signaling. All
protocadherins are expressed mainly in the brain and likely contribute to cellular diversity through alternative splicing, monoallelic
expression regulation, and various combinations of heterodimerization [Murata et al., 2004; Esumi et al., 2005; Hulpiau and van
Roy, 2009]. They are active during brain development and are
considered crucial to human brain function [Blanco et al., 2000;
Dibbens et al., 2008].
PCDH11X/Y is a member of the non-clustered, protocadherind1 subgroup of the cadherin superfamily. It is composed of coding
exons 1 and 2, which contain the extracellular cadherin repeats, the
transmembrane domain and the 50 part of the cytoplasmic tail. Up
to 5 additional, alternatively spliced exons contain the conserved
motifs within the cytoplasmic domain. The 30 end of exon 2 is highly
conserved and the transcripts ending at this point arise through
readthrough of intron 2, although one alternative transcript makes
use of a stop codon introduced by the alternatively spliced exon 3a.
It has been proposed that the short transcripts produce truncated
proteins that are dominant-negative in opposition to the activity of
the longer isoforms [Vanhalst et al., 2005].
PCDH11Y is absent in non-human hominoid species [Durand
et al., 2006]. An ancestral duplicative translocation of the X-linked
PCDH11X to the Y chromosome and subsequent sequence modifications to both genes have been proposed to be the accelerant for
evolution of human cerebral asymmetry, psychosis and language
development [Crow, 2002, 2008; Williams et al., 2006]. Both genes
are expressed in the human brain, most highly in the cortex
[Durand et al., 2006], however their patterns of expression differ
[Blanco et al., 2000]. The PCDH11X/Y gene pair is subject to
complex patterns of alternative splicing in regions of the brain
that support the hypothesis that they play an important role in the
brain related to language and thinking [Ahn et al., 2010].
To date, only a few genes have been directly linked to the ability of
humans to learn language, and these have been discovered through
case studies of families and individuals with language impairment.
A mutation in the transcription factor, FOXP2, was found to be
responsible for the dominant inheritance pattern of a Specific
Language Impairment (SLI) disorder in one family [Lai et al.,
2001] and mutations in CNTNAP2, a target of FOXP2, were found
in a group of Amish children with, among other deficits, language
regression [Strauss et al., 2006]. Although the PCDH11X/Y gene
pair has been previously suggested as candidate for a number of
SPEEVAK AND FARRELL
487
FIG. 2. FISH using BAC probe RP11-339M12 (green) and Xq telomere (red). A: Mother, showing one X chromosome deleted at Xq21.31. B: Proband,
showing deletion at Xq21.31 and Yp11.2. C: Father, showing normal hybridization at Xq21.31 and Yp11.2.
neurological disorders, no direct evidence supporting this has been
found thus far. Sequence variation in PCDH11X/Y was studied
previously in order to investigate the possibility that the gene pair is
etiologically important in psychiatric disorders [Giouzeli et al.,
2004; Durand et al., 2006]. This hypothesis arose due to the finding
that individuals with duplication of the X or Y chromosomes have
an increased chance of developing schizophrenia or other behavioral disorders [Yoshitsugu et al., 2003; Mulligan et al., 2008]. It was
also suggested that male vulnerability to autism, attention deficit
disorder and other abnormal behaviors could be attributable to
variation in the PCDH11Y gene [Kopsida et al., 2009]. To date, no
evidence has been found showing that the PCDH11X/Y gene pair is
important in these disorders. However, single nucleotide polymorphism (SNP) variants only were analyzed in these studies.
Recently, a small familial duplication of 182 Kb within intron 2 of
PCDH11X was described [Whibley et al., 2010]. The family consisted of two males with the duplication and who had an intellectual
disability. However, the duplication was absent in a mildly affected
sister; thus a causal relationship could not be proven. In the case
presented here, a similarly sized, intron 2 deletion in PCDH11X was
inherited from an unaffected carrier mother. The PCDH11Y intron
2 deletion was found to be de novo in this child, which was an
FIG. 3. Screenshot showing the position (highlighted) of the BAC probe, RP11-339M12, within intron 2 of PCDH11X (top) and PCDH11Y (bottom),
using Alamut (Interactive Biosoftware).
488
unexpected finding. It is possible that the PCDH11Y deletion was a
spontaneous, independent meiotic event in the male gamete. An
alternative explanation is that the Y chromosome deletion was the
result of an early, post-zygotic gene conversion event involving the
already deleted maternal PCDHY11X gene. Gene conversion events
are known to occur outside the pseudoautosomal region of the
X and Y chromosomes [Rosser et al., 2009] and a mitotic gene
conversion resulting in a homozygous mutation of an autosomal
gene was reported previously [Zaragoza et al., 2005].
Although the PCDH11X/Y deletions appear to be restricted to
a non-coding region, it is possible that they interfered with the
normal splice mechanisms of the genes, and thus impacted upon
gene expression and the child’s facility to learn language. We are
unable to test for this, since RNA was not collected from the patient,
and because the brain is the main tissue of expression for the
different isoforms of these genes.
De novo copy number changes (CNCs) are suspected of having a
contributory role in sporadic neuropsychiatric disorders, including
autism spectrum disorder and schizophrenia [Lakshmi et al., 2007;
Xu et al., 2008]. The mutation rate of de novo copy number changes
of less than 500 Kb in size in this population is 3–10 times the rate
seen in unaffected controls [Lakshmi et al., 2007; Itsara et al., 2010].
Additionally, rare copy number variations, and in particular, gene
encompassing deletions, have been implicated in autism spectrum
disorders, which include impairment of communication [Pinto
et al., 2010]. This case represents in vivo evidence supporting the
hypothesis that the PCDH11X/Y ancestral gene pair contributed to
the development of speech in humans, and that rare copy number
variation in this gene may be a mechanism for SLI disorders.
However, since de novo CNCs may occur without clinical phenotype, new cases of PCDH11X/Y CNCs in families and children with
neurological deficits are needed to confirm our finding.
ACKNOWLEDGMENTS
We thank Elzbieta Furgala for the FISH analyses and Chantal
Murray for the molecular analyses.
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