Non-syndromic language delay in a child with disruption in the Protocadherin11XY gene pair.код для вставкиСкачать
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: firstname.lastname@example.org 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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