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Int. J. Devl Neuroscience 20 (2002) 407–419

22q11 DS: genomic mechanisms and gene function inDiGeorge/velocardiofacial syndrome

Thomas M. Maynarda,b, Gloria T. Haskella,c, Jeffrey A. Liebermand,Anthony-Samuel LaMantiaa,c,∗

a Department of Cell and Molecular Physiology, CB #7545, UNC School of Medicine, University of North Carolina, Chapel Hill, NC 27599, USAb UNC Neurodevelopmental Disorders Research Center, UNC School of Medicine, University of North Carolina, Chapel Hill, NC 27599, USA

c UNC Neuroscience Center, UNC School of Medicine, University of North Carolina, Chapel Hill, NC 27599, USAd Department of Psychiatry, UNC School of Medicine, University of North Carolina, Chapel Hill, NC 27599, USA

Received 7 February 2002; received in revised form 12 April 2002; accepted 15 April 2002

Abstract

22q11 deletion syndrome (22qDS), also known as DiGeorge or velocardiofacial syndrome (DGS/VCFS), is a relatively common geneticanomaly that results in malformations of the heart, face and limbs. In addition, patients with 22qDS are at significant risk for psychiatricdisorders as well, with one in four developing schizophrenia, and one in six developing major depressive disorders. Like several otherdeletion syndromes associated with psychiatric or cognitive problems, it has been difficult to determine which of the specific genes in thisgenomic region may mediate the syndrome. For example, patients with different genomic deletions within the 22q11 region have beenfound that have similar phenotypes, even though their deletions do not compromise the same set of genes. In this review, we discuss theindividual genes found in the region of 22q11 that is commonly deleted in 22qDS patients, and the potential roles each of these genes mayplay in the syndrome. Although many of these genes are interesting candidates by themselves, we hypothesize that the full spectrum ofanomalies associated with 22qDS may result from the combined result of disruptions to numerous genes within the region that are involvedin similar developmental or cellular processes.© 2002 ISDN. Published by Elsevier Science Ltd. All rights reserved.

Keywords: 22q11 DS; Genome; Gene function

1. Introduction

22q11 deletion syndrome (22qDS, OMIM 192430) isa relatively common genetic anomaly, affecting, approxi-mately 1 in 4000 live births (Devriendt et al., 1998). Pa-tients with 22qDS have a distinctive set of developmentaldefects, including anomalies in craniofacial patterning (e.g.wide set eyes, high nasal bridge), limb and digit anomalies,and cardiac malformations (Driscoll, 1994; Driscoll et al.,1992; Wilson et al., 1991, 1992). In addition to its phys-ical manifestations, 22qDS is associated with behavioraland psychiatric complications: approximately one in fouraffected patients develop schizophrenia, while another onein six develop major depressive disorders (Murphy et al.,1999). Because of its association with schizophrenia anddepression, 22qDS has attracted attention as a model for

∗ Corresponding author. Tel.:+1-919-966-1290.E-mail address: [email protected] (A.-S. LaMantia).

investigating the genetic and developmental neurobiologicalbasis of mental illness.

2. The complex genetics of neurodevelopmentaldisorders

Schizophrenia, like many other psychiatric disorders, ap-pears to have numerous genetic and non-genetic components(seeMaynard et al., 2001, for review). The OMIM onlinedatabase of genetic information lists around 15 independentgenetic loci that have been linked to schizophrenia, althoughnone of these genetic factors can account for all cases ofschizophrenia. Thus, several of these factors are only closelyassociated with schizophrenia risk in one population group,but are unlinked in other populations (de Chaldee et al.,2001; Lachman et al., 1998; Liou et al., 2001; Wei andHemmings, 1999). The linkage between 22q11 deletions andschizophrenia, however, is relatively robust, as a high pro-portion of deleted patients have been observed to develop

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schizophrenia in several independent studies (Bassett andChow, 1999). While genetic factors may significantly in-crease one’s risk of developing schizophrenia, genetics mayonly be part of the cause: the genetically identical twin ofa schizophrenic patient has a 50% chance of developingschizophrenia as well (Davis et al., 1995).

As the correct formation and function of neural circuitsinvolves a myriad of genes—from structural proteins to sig-naling molecules—it might be expected that interrupting thefunction of any of these genes could affect brain function.What makes the interpretation even more challenging is thatmany of the genetic risk factors associated with psychiatricconditions are not even mapped to single genes, but to largersegments of the genome, such as is the case for 22qDS.As outlined in Table 1, numerous genetic syndromes re-sult from the heterozygous deletion (haploinsufficiency) orduplication (trisomy) of segments of chromosomes, ratherthan a mutation within a single gene. Many of these dele-tion/duplication syndromes are associated with mental re-tardation, but others are associated with psychiatric disor-ders such as schizophrenia, epilepsy, autism, and behavioraldisorders. Although these genetic anomalies may provideinsight to the underlying causes of complex behaviors, thestudy of these disorders at the molecular and cellular levelhas proven to be quite challenging.

3. Deletions and duplications: a frequent sourceof neurodevelopmental disorders

While some genetic conditions are limited to singlegenes, most de novo genetic anomalies involve more com-plex rearrangements of chromosomes involving numerousgenes. This category of chromosomal anomalies, includingdeletions, duplications or translocations of chromosomal

Fig. 1. Mechanism of LCR mediated deletions. As described byEdelmann et al., 1999a,band Shaikh et al. (2001), recombination between mismatchedhomologous elements (LCRs) can lead to the deletion of chromosomal segments. Mismatches can occur within a single chromosomal segment (A or C),leading to a excision in one chromosome, while the other chromosome is intact. Alternatively, mismatches between LCRs on both chromosomal pairscan lead to a deletion in one (C and D) chromosome, with a reciprocal duplication in its chromosomal pair. While most patients (∼85%) have largedeletions mediated by the most distal and proximal LCRs (A and B), a smaller number have atypical deletions mediated by the medial LCR (C and D).

segments can be found in approximately 1 in 165 newborninfants, and in 1 in 4 miscarriages and stillbirths (Shafferand Lupski, 2000). Although some of the syndromes result-ing from these rearrangements are quite rare, others occurquite frequently. 22q11 DS is relatively common, affectingaround 1 in 4000 newborns, which is only slightly lesscommon than Down syndrome, the most common geneticbirth defect, which affects approximately 1 in 1000 livebirths (Hook et al., 1983).

Because the incidence of these genetic rearrangementsvary dramatically depending on the chromosomal region in-volved, it has long been suspected that the genomic structureof certain chromosomal regions may be vulnerable. Whilethe mechanism for many of these rearrangements is notyet known, the probable cause for deletions, duplications,and rearrangements for others is becoming clear. For exam-ple, several studies have illustrated that the most commondeletions observed within the 22qDS population occur be-tween sets of repeated elements known as low copy repeats(LCRs). Within the region of 22q11, there are several setsof LCRs, which are genomic elements, of approximately200 kb in length that share high homology with each other(Edelmann et al., 1999a,b; Shaikh et al., 2000, 2001). Dur-ing gametogenesis, the normal process of homologous re-combination results in the alignment and recombination ofpaternal and maternal chromosomal material. Because theseLCRs share high homology with each other, it appears thatwithin the 22q11 region, the wrong LCRs can align inap-propriately during recombination events, leading to a dele-tion on one recombinant chromosome and a duplication onthe other (seeFig. 1). This unequal crossing over appears tobe a common mechanism that leads to similar deletions andduplications at some other chromosomal sites, and may besimilarly involved in some translocations between chromo-somes (Shaffer and Lupski, 2000).

410 T.M. Maynard et al. / Int. J. Devl Neuroscience 20 (2002) 407–419

4. The variability of 22q11 deletion syndrome

Throughout its history, 22qDS has been called by manynames, including DiGeorge syndrome (DGS), Shprintzensyndrome, velocardiofacial syndrome (VCFS), conotrun-cal anomaly face syndrome, and CATCH22 (cardiac ab-normality, abnormal face, T cell deficits, cleft palate andhypocalcemia). This diversity of names reflects the variablephenotype that results from 22q11 deletions. Because theseverity of each of the individual symptoms varies signifi-cantly, patients with 22qDS were often classified differentlybased on the relative severity of each of their symptomsbefore the common genetic mechanism that underlies thedisorder was understood.

The symptoms presented by 22qDS patients can bequite variable, however, there is much less heterogeneityat the genetic level. Although there are four LCRs withinthe 22q11 region, the majority (∼87%) of 22qDS patientshave essentially identical 3 Mb deletions, removing the seg-ment between the most proximal and distal LCRs (Shaikhet al., 2000). A smaller subset of patients (∼10%) haveintermediate 1.5–2 Mb deletions that include the other twoLCRs, while an even smaller number have “atypical” dele-tions or translocations that also result in the symptoms ofVCFS/DGS (Amati et al., 1999; Shaikh et al., 2000, 2001).While the majority of 22qDS patients have similar 3 Mbdeletions, a similar set of physical and psychiatric symptomshave been observed among the remaining 13% of patientswith smaller or atypical deletions. Indeed, a handful ofpatients have even been identified with different balancedtranslocations within 22q11 that lead to the VCFS/DGSphenotype (Couillin et al., 1992; Demczuk et al., 1995;Sutherland et al., 1996; Wadey et al., 1995). In light of suchevidence, it does not appear likely that the severity or rangeof symptoms in 22qDS patients can be directly associatedwith the disrupted expression of a single gene within 22q11.

Another source of variability that has been noted withother deletion/duplication syndromes arises from genomicimprinting. In some cases, the genomic material contributedby the maternal or paternal gamete are not functionallyequivalent, and similar chromosomal anomalies can resultin a dramatically different phenotype. The strongest evi-dence for this phenomenon can be found in studies of thedeletion of chromosome 15q11, leading to Prader–Willi syn-drome (OMIM 176270) and Angelman syndrome (OMIM105830). Offspring that inherit a deletion on the paternalchromosome display modest limb and craniofacial dys-morphologies, and mild to modest mental retardation andbehavioral problems characteristics of Prader–Willi syn-drome. If the same deletion is inherited from a maternalchromosome, however, the effects are much more severe,and include severe retardation and epilepsy. The source ofthis phenomenon, genetic imprinting, is due to the differ-ent transcriptional activities of maternally and paternallyderived genes (Hanel and Wevrick, 2001). At least 1% ofhuman genes may have some level of imprinting (Hanel and

Wevrick, 2001), and the effects of a deletion may dependsignificantly on whether the remaining chromosome is im-printed. In the case of 22qDS, the evidence for imprinting isambiguous. Some studies have suggested that the proportionof 22qDS patients with maternal and paternal deletions areessentially the same (Fokstuen et al., 1998), suggesting thatimprinting may not be a major component of the disorder.However, another study suggests that imprinting may be afactor in at least one aspect of the syndrome: high resolutionMRI brain images have demonstrated that VCFS patientsshow reduced gray matter volume when compared withcontrol subjects, but patients with maternally derived dele-tions have even greater brain volume reductions than thosewith paternally derived deletions (Eliez et al., 2001). Thus,although the parental origin of the deletion is not the solearbiter of the 22qDS phenotype, imprinting may accountfor at least part of the variability of the syndrome. Althoughthe source, size, and position of the deletion may determineat least some degree of variability, it is possible that thesyndrome is inherently variable. Studies of monozygotictwins with 22q11 deletions have demonstrated that identi-cal deletions do not ensure identical phenotypes (Goodshipet al., 1995; Vincent et al., 1999). As with schizophreniaand other complex genetic disorders, genotype alone cannotentirely predict the outcome of 22q11 deletions.

5. The neurodevelopmental basis of 22qDS

One insight that may prove useful in the analysis of thedeletion/duplication syndromes, and add to their usefulnessas models for psychiatric disorders, is that their roots maylie in the disruption of normal neural development. Whileevidence for the developmental basis of the most severeconditions has long been obvious (such as the severe birthdefects associated with the profound retardation in Downsyndrome) a similar set of developmental anomalies is alsoassociated with the genetic syndromes that serve as mod-els for many psychiatric disorders. As outlined inTable 1,most of the deletion/duplication syndromes are associatedwith mild to severe anomalies, often in craniofacial, limband heart development. The development of each of thesestructures involves a mechanism called inductive signaling,where interactions between embryonic tissues leads to thepatterning and formation of mature structures. Thus, in theembryo, interactions between mesenchymal and epithelialtissues in the limb buds, branchial arches, and aortic arches(Fig. 2A) are essential for the proper development of themature limbs, craniofacial structures, and the outflow tractof the heart, respectively. In addition, the same inductivesignaling mechanism that underlies the patterning of limbs,hearts, and faces is also used in the patterning of the ner-vous system, particularly in the forebrain (LaMantia et al.,2000). Because the same molecular signals that patternlimbs, hearts, and faces also pattern the nervous system, it islikely that obvious physical anomalies may be evidence of


Fig. 2. Sites of mesenchymal/epithelial inductive signaling in the mouseembryo. In the developing forebrain and frontonasal process, as well asin the branchial arches, limb buds, and aortic arch, signaling interactionsbetween epithelial tissues and mesenchymal cells lead to the patterningand organization of the embryo (A). As developmental disruptions to eachof these sites is implicated by the limb, heart, and craniofacial anomalies,as well as the cognitive and psychiatric symptoms observed in 22qDSpatients, these patterning processes may be interrupted by some aspectsof the syndrome. Several 22q11 gene homologues are expressed at eachof these sites in the developing mouse embryo, including RanBP1 (B)and DGCR2 (C).

subtle brain anomalies that may be more difficult to detect(Maynard et al., 2001).

The linkage between physical defects and psychiatric con-ditions is not only limited to any specific “genetic model”—indeed, even in the absence of a detectable genetic anomaly,a significant proportion of patients with schizophrenia havedetectable physical anomalies (Guy et al., 1983), and thesemay be related to subtle changes in the brain (Turetsky et al.,2000). Although it is not entirely clear what types of phys-ical changes within the brain contribute to schizophrenia,there is growing evidence that specific neural circuits are of-ten disrupted in schizophrenic patients (Glantz and Lewis,2000; Selemon and Goldman-Rakic, 1999; Selemon et al.,1998). In a similar fashion, disruptions in neural circuits in-volving the dendrites and spines of brain neurons have alsobeen reported in other genetically based neurological andpsychiatric conditions (Kaufmann and Moser, 2000). Thus,

it appears that the deletion syndromes may not only haveexternal similarities (such as limb and craniofacial anoma-lies), but they may also share some common basis in thebrain, in the form of compromised neural circuitry.

The inductive patterning of the limbs, face, and aorticarches of the heart are all influenced by a population of mes-enchymal cells derived from the neural crest. Early in devel-opment, neural crest cells migrate from the dorsal portion ofthe developing neural tube, and migrate into positions adja-cent to each of these developing structures (Noden, 1988).The developmental anomalies that characterize 22qDS canbe observed in each of these structures, in the form of cran-iofacial, limb and digit anomalies, and defects in the aorticarches. Thus, it is thought that at least some aspects of22qDS may result from abnormal neural crest cell migrationor function (Epstein, 2001; Maynard et al., 2001; Scambler,2000). Because a similar population of neural crest alsopatterns the early development of the forebrain, disruptionsto this mechanism may also lead to disruptions in the brainas well (LaMantia et al., 2000; LaMantia, 1999), althoughthe exact brain deficits involved in 22qDS are not yetwell understood. Accordingly, attention has turned to theanalysis of the expression pattern of 22qDS genes duringembryonic development, to determine which of these genesmay be present during the development of the neural crest,or in tissues whose patterning is mediated by mesenchy-mal/epithelial interactions involving crest-derived cells.

6. A closer look: the functions of the 22q11 genes

Although the majority of patients with 22q11 deletionshave similar 3 Mb deletions (Shaikh et al., 2000), most atten-tion has been focused on the 1.5 Mb region between the twomost proximal LCRs (Fig. 3). Roughly 1 in 12 patients havethis shorter deletion, and the symptoms observed in patientswith this deletion are indistinguishable from those with thelarger deletion. Thus, it is presumed that this region is funda-mental to the phenotype. Approximately 30 genes are con-tained within this 1.5 Mb DiGeorge critical region (DGCR).Because 22qDS patients display a characteristic set of de-velopmental anomalies in their limbs, hearts, and craniofa-cial structures, in addition to characteristic neurological andpsychological deficits, it is expected that the gene or genesresponsible for the condition will likely function during thedevelopment or maturation of these structures. As detailedbelow, the expression pattern and potential function of manyof the 22q11 genes have been examined, and numerous can-didates have arisen that are worthy of further consideration.

7. Transcription factors

Although there are a diverse set of genes within the 22q11region with a variety of potential functions, one subset ofgenes has been closely investigated for its potential role


Fig. 3. Map of 22q region. Left: map of chromosome 22q11, indicatingposition of several genetic landmarks. Approximate position of the threeLCRs that mediate the common deletions are indicated by dashed lines.The top two LCRs are separated by approximately 1.5 Mb, while thelower LCR is approximately 3 Mb from the top one. Right: map of genesfound within the DGCR defined by the 1.5 Mb deletion.

in mediating the phenotype of the syndrome. Transcriptionfactors—proteins with DNA binding elements that are capa-ble of regulating the expression of large subsets of genes—could be responsible for coordinating the morphogeneticprograms that lead to the development of embryonic struc-tures. For this reason, four putative transcription factorswithin the DGCR were among the first genes that were char-acterized as potential mediators of the 22qDS phenotype.

The first of these transcription factors, HIRA (also calledTuple1), encodes a nuclear protein with histone-bindingproperties that have been conserved from yeast to humans.Two HIRA homologues in yeast, Hir1p and Hir2p, are tran-scriptional corepressors working at the chromatin level in acell-cycle regulated manner. Similar to Hir1p, HIRA con-tains several elements called WD repeats that are commonlyfound in transcriptional control proteins (Halford et al.,1993a; Lamour et al., 1995; Scamps et al., 1996), and isthought to function as part of a multiprotein complex. Sev-eral HIRA binding proteins have also been described, in-cluding Pax3, a homeodomain protein critical for patterning

and embryogenesis (Magnaghi et al., 1998). HIRA, like itsyeast homologs, may also function as a cell-cycle regulatedtranscriptional repressor. During mitosis, HIRA appears tobe modified by a cell-cycle dependent kinase, causing itto lose its affinity for histone-bound chromatin (De Luciaet al., 2001), and instead bind directly to DNA itself. Asa consequence, when HIRA is over-expressed in dividingcells, it causes the cell cycle to stall in S phase (Hall et al.,2001). HIRA is expressed in the mesenchyme of the de-veloping limbs, craniofacial structures, and heart (Wilminget al., 1997), and as such, it could be essential for thedevelopment of these structures. Consistent with this, ex-periments that blocked the function of HIRA in the trunk ofdeveloping chicken embryos led to an increased incidenceof persistent truncus arteriosis, similar to that observed inmany human patients (Farrell et al., 1999).

Another transcription factor, Tbx1, has recently gainedattention as a potential mediator of the aortic arch defectsseen in 22qDS. Tbx1 is a member of a gene family that con-tains a DNA-binding domain (called the “T-box”) similar tothat contained by the mouse Brachyury (T) gene. The mouseand human Tbx1 genes display a similar expression patternduring early embryogenesis, most prominent in the pharyn-geal arches and pouches and in the otic vesicle (Chapmanet al., 1996; Chieffo et al., 1997). Later in development,Tbx1 is seen in the vertebral column and tooth bud. Tbx1is required for the proper development of the heart, and itshaploinsufficiency in mice causes aortic arch defects similar,but not entirely identical to those seen in human VCFS/DGSpatients. However, the heart defects only appear in a smallproportion of haploinsufficient mice, possibly because othermechanisms may allow for repair and recovery of disruptedarch development (Lindsay and Baldini, 2001). In addition,the complete loss of Tbx1 in the mouse results in devel-opmental defects that more closely approximate 22qDS, in-cluding hypoplasia of the thymus and parathyroid glands,cardiac outflow tract anomalies, and some craniofacial andskeletal defects (Jerome and Papaioannou, 2001; Lindsayet al., 2001; Merscher et al., 2001). However, even in micecompletely lacking Tbx1, a significant subset of mice stilldevelop normally. Expression of Tbx1 in the mouse pharyn-geal arch is dependent on Shh signaling, a developmentallyregulated pathway critical for the proper patterning and sub-sequent morphogenesis of several non-axial structures, in-cluding the heart (Garg et al., 2001). Shh is thought to actearly in the process of morphogenesis by setting up regionalboundaries of gene expression. Apparently, Tbx1 can func-tion downstream of Shh to affect pharyngeal arch forma-tion. Although Tbx1 haploinsufficiency is likely to be a ma-jor determinant of aortic arch malformations in people with22qDS, no mutations that disrupt the function of Tbx1 havebeen described in patients who have the symptoms of 22qDSbut lack a detectable deletion (Gong et al., 2001). BecauseTbx1 does not appear to function in the developing nervoussystem, it is unlikely that disruption of this one gene canaccount for the cognitive and psychiatric aspects of 22qDS.


Another transcription factor, E2F6, the most recently iden-tified member of the E2F family of transcription factors(Kherrouche et al., 2001), is likely involved in regulationof the cell cycle. Two splice variants of E2F6 exist, andboth forms are expressed during mouse embryogenesis aswell as in the adult mouse. The level of E2F6 increasesrapidly during the initiation of the cell cycle, and overex-pression of E2F family members alters cellular prolifera-tion (Kherrouche et al., 2001; Wang et al., 2001). Func-tional studies suggest that the biological properties of E2F6are mediated via its ability to recruit other regulatory pro-teins, such as those in the polycomb complex (Trimarchiet al., 2001). Two genes in the DGCR share a potential E2Fbinding site—RanBP1 (Htf9a) and Htf9c. Both genes aredivergently transcribed from a shared promoter, whose ac-tivity may be regulated by cell cycle progression (Di Fioreet al., 1999; Guarguaglini et al., 1997). Regulated activityof RanBP1 is required for the organization and function ofthe mitotic spindle in mammalian cells (Guarguaglini et al.,2000). RanBP1 activity is also required for receptor me-diated nucleocytoplasmic transport, a fundamental cellularpathway responsible for shuttling proteins from the cyto-plasm to the nucleus (Kunzler et al., 2001). In the developingmouse, RanBP1 is expressed in the frontonasal mass, heart,limb buds, and branchial arches, as well as the neural tube(Maynard et al., 2002; see alsoFig. 2B). Htf9c shares thesame promoter as RanBP1, and likely shares the same de-velopmental expression pattern and cell cycle-regulated ex-pression. Although Htf9c encodes a protein with similarityto some yeast and bacterial nucleic acid modifying enzymes,its actual function remains unknown (Bressan et al., 1991).

Another transcription factor, GSCL, is a homeobox generelated to Goosecoid, a gene required for proper cranio-facial development. GSCL is expressed during early braindevelopment, primarily in a subregion of the pons, and isalso expressed in the gonad and the gut as well (Gottliebet al., 1997). Interactions between GSCL and other devel-opmental proteins have been described (Galili et al., 2000),although the function(s) of GSCL remain elusive. The ex-pression of GSCL may be associated with, or even influ-ence, that of another gene in the DGCR—DGCR1, alsoknown as Es2 (Lindsay et al., 1998). The expression of thetwo genes partially overlap in the developing pons, and inserotonin-positive cells of the raphe nuclei. DGCR1 expres-sion in the pons is lost in gscl null mice, which otherwise ap-pear completely normal (Wakamiya et al., 1998). DGCR1, aprotein of unknown function, has homologues inC. elegansandDrosophila, both of unknown function as well. DGCR1is widely expressed during embryogenesis, with higher ex-pression in the nervous system.

8. Cell cycle and signaling

Another candidate gene, Cdc45L, is so named for itshomology to a yeast cell-cycle regulatory protein that is

required for the initiation of DNA replication. The vertebrateCdc45L seems to play a similar role, as studies suggest that itbinds to and recruits members of the complex of proteins in-volved in the initiation of DNA replication (Kukimoto et al.,1999; Mimura and Takisawa, 1998). Mice lacking Cdc45Ldisplay impaired proliferation of the inner cell mass anddie shortly after implantation, although heterozygotes liveto adulthood without apparent abnormalities (Yoshida et al.,2001). The highest levels of Cdc45L expression are seen inadult testis and thymus, and in fetal liver, although transcriptscan also be detected in fetal brain, branchial arches, and kid-ney, all of which are affected in 22qDS (Shaikh et al., 1999).

Another gene with homology to yeast cell cycle pro-teins, CDCrel-1 (also known as PNUTL1), is related to afamily of proteins called septins. In yeast, these proteinsare thought to be signaling molecules involved in cytoki-nesis. In vertebrates, septins likely have a similar role,although expression data suggests they may have additionalfunctions. In the mouse embryo, CDCrel-1 is expressed indorsal root ganglion neurons, cranial ganglia, and the lat-eral layer of the neural tube where terminally differentiatedneurons are found. At later stages, it is expressed in themesenchyme of both the frontonasal mass and limb buds(Maldonado-Saldivia et al., 2000). Additionally, CDCrel-1is expressed in the mature nervous system in the termi-nals of axons, where it may play a role in neurotransmitterrelease (Beites et al., 1999; Toda et al., 2000). AlthoughCDCrel-1 may have important functions in synaptic regu-lation, mice lacking CDCrel-1 appear normal, as do theirneurons and synapses. However, the expression patternsof related septin molecules are changed when CDCrel-1 isremoved, and these other septins may possibly subsititutefor its loss (Peng et al., 2002).

Recent studies indicate that CDCrel-1 may have twoforms, which are differentially expressed during develop-ment and in maturity (Toda et al., 2000). Interestingly, bothforms appear to contain within them the sequence for an-other 22qDS gene, GPIb�, which resides just downstreamof CDCrel-1 on the genome (Kelly et al., 1994; Yagi et al.,1994). GPIb� has been well studied as one of four subunitswhich together constitute a functional receptor complex forthe von Willebrand factor, which is important for plateletfunction. This receptor complex is involved in platelet ad-hesion, activation, and aggregation. It is therefore possiblethat disruptions to GPIb� may be lead to disruptions in theformation or function of platelets, a symptom that has beenobserved in some schizophrenic 22qDS patients (Lazieret al., 2001).

Ufd1L is named for its homology to the yeast ubiquitinfusion-degradation protein, an essential component for yeastprotein and mRNA processing. Ufd1L may play a similarrole in regulating protein processing in humans and mice aswell (Botta et al., 2001). Ufd1L gathered particular interestas a candidate gene after a 22qDS patient was identifiedwith a small 20kb deletion that selectively disrupted theUfd1L gene (Yamagishi et al., 1999). In addition, a single


nucleotide polymorphism in the promoter region of Ufd1Lhas been linked to schizophrenia in some studies (De Lucaet al., 2001). Like several other DGCR genes, Ufd1L isexpressed in the limb buds, heart, and branchial arches ofthe developing mouse embryo, and is also expressed in themedial telencephalon of the developing brain. Like RanBP1,the Ufd1L gene has a bidirectional promoter that is alsoshared by a second 22q11 gene, Cdc45L (Kunte et al., 2001).

A diverse set of potential signaling molecules are alsofound in the DGCR. Most of these genes remain largely un-characterized. One of these genes, ARVCF, is named for its10 repeated elements that are similar to ones found in theDrosophila protein armadillo (Sirotkin et al., 1997b). Thesuggested function of ARVCF is at adherens junctions, and islikely similar to the function of the related�-catenin protein,which is involved in cell signaling through the wnt family ofligands and receptors (Kaufmann et al., 2000). Expressionanalysis shows that ARVCF is present in multiple fetal andadult tissues. (Sirotkin et al., 1997b). NLVCF is a novel geneof unknown function that contains two consensus nuclearlocalization signals (Funke et al., 1998). NLVCF is mappedbetween HIRA and Ufd1L, and it has been suggested thatthe expression of NLVCF and HIRA are transcribed in op-posite directions from the same promoter region, and as aconsequence both may share some of the same regulatory el-ements. In situ hybridization analysis reveals that NLVCF isexpressed in most structures of an early mouse embryo, withespecially high expression in the head, as well as the firstand second branchial arches (Funke et al., 1998). Similarly,T10 encodes a small protein of unknown function with noapparent homologies to previously described genes (Halfordet al., 1993b). The predicted T10 protein is rich in serineand threonine residues, and its activity may be regulated byphosphorylation. T10 is expressed in the developing mouseembryo at various stages, including some structures affectedin 22qDS, such as the face and heart. Two other putativesignaling molecules have also been localized to the typicallydeleted region, Tsk1 and Tsk2 (Goldmuntz et al., 1997).These small intronless genes encode serine-threonine ki-nases that appear to be primarily expressed in testes (Nayaket al., 1998).

Another signaling molecule, BCR, is widely known as onehalf of the BCR–ABL fusion oncoprotein responsible formany forms of chronic myelogenous leukemia (Sattler andGriffin, 2001). Numerous patients with a t(9;22) chromoso-mal translocation have been found where the c-Abl gene ofchromosome 9 fuses with the BCR gene of chromsome 22.The result of this fusion, the BCR–ABL protein, acts as anactivated form of ABL, and results in unregulated cell prolif-eration. BCR itself functions as a component of the signalingcascade involving the Rho GTPase protein (Chuang et al.,1995). BCR also appears to be involved in the modulation ofbehavior, as mice lacking BCR exhibit defects in their hor-monal and stress responses. As a consequence, when micelacking BCR are exposed to physiological or social stress,they display prolonged elevation of plasma glucocorticoids

or increased male aggression, respectively (Voncken et al.,1998).

9. Cell adhesion molecules

Several other genes have been mapped to the DGCR, butremain relatively uncharacterized. Three of these—DGCR2,DGCR6, and TMVCF—may have roles in regulating cel-lular adhesion. DGCR6 encodes a protein that shares somesimilarities with gonadal (gdl), aDrosophila gene of un-known function, and to the gamma chain of laminin, acomponent of the extracellular matrix (Demczuk et al.,1996). Like gdl, DGCR6 contains a predicted coiled-coildomain, which is a structure that is thought to be involvedin protein–protein interactions. Northern blot analysisshows that DGCR6 is expressed in the developing mousefrom early embryogenesis onward, as well as in severaladult structures including the nervous system (Lindsay andBaldini, 1997). Apparently, two functional copies of thegene are present on human 22q11 due to a duplicationevent over 12 million years ago; however, both are usuallydeleted in 22qDS patients (Edelmann et al., 2001).

DGCR2, another gene of unknown function, shares sig-nificant homology with a protein called Sez-12. Sez-12has been described as a membrane glycoprotein that wasoriginally identified in a screen for seizure-sensitive genes.DGCR2 and Sez-12 are 99% identical at the amino acidlevel; Indeed, they may be the same gene (Kajiwara et al.,1996; Taylor et al., 1997). Structural analysis of the geneshows that, in addition to other well-described domains,DGCR2 contains a domain that is similar to one found ina developmentally important regulatory gene called Msx2.DGCR2 is widely expressed early in mouse embryogenesis,with strong expression seen in the frontonasal mass, thebranchial arches, the limb buds (forelimb bud in particular),and somites (Taylor et al., 1997; see alsoMaynard et al.,2002andFig. 3).

TMVCF, also known as claudin-5, is a member of theclaudin family of tight junction proteins (Morita et al.,1999a,b; Sirotkin et al., 1997a). TMVCF is expressed inmany tissues, but appears to be specifically localized tocell-cell borders of endothelial cells. The protein sequenceis small (219 AA’s) with two membrane spanning domains.TMVCF is expressed early in the developing mouse, andin several adult structures as well, including human lung,heart, and skeletal muscle.

10. Metabolic cofactors and enzymes

A small number of metabolic or housekeeping genes havealso been localized to the typically deleted region. While itis not known which tissues express each of these genes, it islikely that they participate in multiple functions in a varietyof cell types. Gamma glutamyl transferase (GGT) is part of a


large multigene family of enzymes that are involved in manyaspects of cellular metabolism (Morris et al., 1993). Simi-larly, CTP encodes a mitochondrial protein that is involvedin cellular metabolism (Heisterkamp et al., 1995; Stoffelet al., 1996). Finally, thioredoxin-reductase 2 (ThioR2, orTrxR2) encodes a mitochondrial protein also believed to beinvolved in celluar metabolism (Heisterkamp et al., 1995;Stoffel et al., 1996). Thioredoxin reductases catalyze theNADPH-dependent reduction of the thioredoxin protein, aswell as other endogenous and exogenous compounds.

Another metabolic gene, catechol-O- methyl transferase(COMT), encodes a protein that is responsible for the break-down of certain neurotransmitters, catechol hormones, anddrugs such as Levodopa (Grossman et al., 1992; Winqvistet al., 1992). Previous studies indicate a link between alow-activity form of this allele and schizophrenia, particu-larly homicidal behavior (Kotler et al., 1999). In addition, aCOMT theory of 22qDS-associated bipolarity has been pro-posed.Graf et al. (2001)submit that deletion of one COMTallele may reduce the availability of the enzyme, resulting inincreased neurotransmission, which may lead to psychiatricsymptoms.

11. Genes unique to human 22q11

Two other genes, CLTCL and Dvl1L, lie within the1.5 Mb deletion, although they are not present in the mouse.Without a murine model, it is more difficult to assess thefunction and expression of these two genes than it is withthe 22qDS candidate genes. CLTCL encodes a putativeprotein with similarity to the clathrin heavy chain, andmay thus play roles in receptor-mediated endocytosis andsignal transduction (Long et al., 1996). This gene is ofsome interest, as a patient with several features of 22qDSwas described with a balanced translocation interruptingthe 3′ coding region of CLTCL (Holmes et al., 1997). Thesecond gene, Dvl1L, has homology to theDrosophila di-sheveled segment-polarity gene, and is expressed in severalfetal and adult tissues, including the thymus and the heart(Pizzuti et al., 1996). Although theDrosophila disheveledprotein is involved in embryonic patterning, it is not knownwhether this human homologue also has a similar role indevelopment.

12. Uncharacterized and presumednon-functional genes

In addition to the genes described above, there are severalother genes that have been localized to the 1.5 Mb deletionregion. Two of these genes, WDVCF (named for WD-repeatsfound in its coding sequence) and DGCR8, have beenmapped to the region, although no details about their expres-sion or function have been published. In addition, there area handful of other pseudogenes, or non-coding genes such

as DGCR5 that have been mapped to the region, but arepresumed to not have any significant function (Sutherlandet al., 1996). In addition, it is possible that other, as yetundiscovered genes may yet be found within this region.

13. Beyond the minimal DGCR

A handful of other genes that reside within the commonlydeleted 3 Mb region, but outside the 1.5 Mb DGCR haveattracted attention because of their expression patterns andmolecular functions. The first, ZNF74, encodes a presumedtranscription factor that may have a role in RNA process-ing (Aubry et al., 1993). ZNF74 is mainly expressed in thedeveloping neural tube and in neural crest-derived tissues,such as the dorsal root ganglia, sympathetic ganglia, dorsalaorta, and the heart great vessels (Ravassad et al., 1999). An-other gene, Crkl, encodes a protein closely related to the Crkfamily of adaptor proteins. Crk family proteins are widelyexpressed and may mediate some aspects of cell signalingduring growth and differentiation. Mice deficient for Crklexhibit many defects that are reminiscent of 22qDS, suchas anomalies in craniofacial structures, as well as defectsin the cranial ganglia, aortic arch arteries, cardiac outflowtract, thymus, and parathyroid gland (Guris et al., 2001).

Another candidate gene outside of the 1.5 Mb deletionis BID (Footz et al., 1998; Wang et al., 1998). Bid is amember of the Bcl-2 family, and it is believed to promoteapoptotic cell death by inducing the release of cytochrome cand acting as a substrate for the death-activating molecule,caspase 8 (Tafani et al., 2002). BID can apparently mediatecell death under a variety of conditions, including ischemia-induced neuronal cell death, and retinoid-mediated celldeath in tumor cell lines (Ortiz et al., 2001; Plesnila et al.,2001). Programmed cell death is an important aspect ofmany developmental processes, including the developmentof the craniofacial and heart structures, (Graham et al.,1996; Poelmann et al., 2000), although it is not knownwhether BID is involved in these processes.

14. 22qDS: is it all or nothing?

Although the list of 22qDS candidate genes is long andintriguing, there has not been a single candidate gene thathas arisen as the sole cause of the deletion syndrome. Sev-eral candidate genes have attracted attention because of theirexpression pattern, potential function, or because of thephenotype that results from their absence in mouse models.Likewise, studies of human 22qDS patients have revealedsignificant genetic variability, and the diverse array of dele-tions observed in the patient population does not implicatea single gene as the unique cause. The inability to identifya single gene should not be viewed as a failure; rather it isevidence that 22qDS, like many other deletion/duplicationsyndromes, should be classified as a “contiguous gene


syndrome”, as it probably involves numerous genes withinthe region.

If 22qDS is a contiguous gene syndrome, it suggests thatthere must be some form of interaction or relationship be-tween at least a few of the genes in the region. Althoughit is not known whether such relationships exist, there areseveral intriguing potential mechanisms that could involvenumerous 22q11 genes. The first possibility is that severalof the genes may in fact interact directly with one another,perhaps in a regulatory fashion. There is limited evidencefor some potential interactions of this nature; for example,the expression of the RanBP1 and Htf9c genes may be reg-ulated, at least in part, by the E2F6 gene that lies at least1 Mb away within the 22q DGCR. Interactions such as thiscould cause a deletion within one segment of the DGCRto indirectly impact the expression of genes in other, intactsegments as well.

It is also possible that the genetic overlap of the syndromemay not be due to the overlapping molecular functions ofthe genes themselves, but instead to their shared expressionin similar tissues. Thus, although each of the genes involvedmay be molecularly distinct—and involved in completelyunrelated cellular tasks—the set of unrelated genes maybe involved in the same developmental or morphogeneticprocesses. In this manner, even the subtle disruption of adisparate set of genes—including cell signaling genes, tran-scription factors, adhesion proteins, cell cycle regulatoryelements, and even metabolic proteins—could together leadto a significant disruption in the development of structuressuch as the face and the brain. Finally, it is also possiblethat deletions, duplications, or translocations within this re-gion may somehow disrupt some as yet undiscovered levelof regulation, possibly at the chromosomal level.

Acknowledgements

This work was supported by the National Alliance forResearch on Schizophrenia and Depression (NARSAD), aswell as NIH grant HD-40127 to TMM, NIH grant HD-29178to ASL, and MH-33127 to JAL.

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