REVIEWThe 22q11.2 deletion syndrome
Hiroyuki Yamagishi
Department of Pediatrics and Molecular Biology, University of Texas Southwestern Medical Center, TX, USA
Department of Pediatrics, Keio University School of Medicine, Tokyo, Japan(Received for publication on March 27, 2002)

Abstract. The 22q11.2 deletion syndrome (22q11DS) encompasses DiGeorge syndrome, velo-cardiofacial syndrome and conotruncal anomaly face syndrome and is due to a microdeletion of chromosome
22q11.2. This is the most frequent known interstitial deletion found in human with an incidence of 1 in
4,000 live births. A large number of clinical ﬁndings have been reported in affected patients, including
cardiac defects, characteristic facial features, thymic hypoplasia, cleft palate, hypoparathyroidism,
learning difﬁculties and psychiatric disorders. A comprehensive evaluation and follow-up program isnecessary for patients with 22q11DS. A striking aspect of the 22q11DS phenotype is its variability, thebasis of which remains unclear, and no phenotype-genotype correlation has been made. The structures
primarily affected in patients with 22q11DS are derivatives of the embryonic pharyngeal arches andpouches suggesting that haploinsufﬁciency of the gene(s) on the deleted region, spanning 2–3 Mb, isimportant in pharyngeal arch/pouch development. Extensive gene searches have been successful in
identifying more than 30 genes in the deleted segment. Although standard positional cloning has failed
to demonstrate a role for any of these genes in the syndrome, the use of experimental animal models
and advanced genome manipulation technologies in mice have been providing an insight into thedevelopmental role of some of these genes, including TBX1. In this review, the clinical features andmanagement of patients with 22q11DS are integrated with our current understanding of the embryological and molecular basis of this syndrome, as presented at the 1235th Meeting of The Keio Medical
Society. (Keio J Med 51 (2): 77–88, June 2002)
Key words: chromosomal deletion, congenital heart disease, velopharyngeal dysfunction, hypocalcemia, pharyngeal arch development

ism and immune deﬁciency in 1965;5 VCFS was associated with cleft palate, CHD, a distinct facial appearance, and learning difﬁculties in 1978;6 CAFS was
described in 1976 and was characterized by conotruncal
CHD, a distinct facial appearance and hyper-nasal
voice.7 Clinical genetics have gained from the discovery
that these syndromes have overlapping clinical presentations and share a common chromosomal deletion.
DGS, VCFS and CAFS are, therefore, part of 22q11DS
reﬂecting various outcomes of the same underlying genetic defect.
The acronym ‘‘CATCH22 (cardiac defects, abnormal
facies, thymic hypoplasia, cleft palate, hypocalcemia,
and 22q11 deletions)’’ was proposed, in 1993, to aid
in remembering the main features of the syndrome
encompassing DGS, VCFS and CAFS.8 However, clinical use of this term may be inappropriate because of

Introduction
The entity of 22q11.2 deletion syndrome
A heterozygous deletion within chromosome 22q11.2(del.22q11) is the genetic basis of the most common
interstitial deletion syndrome, the 22q11.2 deletion
syndrome (22q11DS) in humans, with an incidence of
1 in 4000–5000 births.1 Most are sporadic in origin,but 10–20% of deletions are inherited as an autosomal
dominant trait. In the early 1990’s, the deletion wasreported in association with three distinct syndromes,
namely DiGeorge syndrome (DGS; OMIM]188400),
velo-cardio-facial syndrome (VCFS; OMIM]192430)
and conotruncal anomaly face syndrome (CAFS;
OMIM]217095).2–4 DGS was originally characterizedby congenital heart defects (CHD), hypoparathyroid-

Presented at the 1235th Meeting of the Keio Medical Society in Tokyo, October 30, 2001.
Reprint request to: Dr. Hiroyuki Yamagishi, Department of Pediatrics and Molecular, Biology, University of Texas Southwestern Medical
Center, 6000 Harry Hines Blvd., Rm. NA8.214, Dallas, TX 75390-9148, USA, e-mail: hyamag@mednet.swmed.edu
77

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Yamagishi H: The 22q11.2 deletion syndrome

following reasons:9 1) The term ‘‘Catch-22’’ has a negative meaning which represents a situation where it is
impossible for you to do anything, originally from Heller’s novel entitled ‘‘CATCH 22’’;10 2) The term ‘‘A’’
referring to ‘‘abnormal facies’’ is difﬁcult to accept forpatients and their family; 3) Recent studies haverevealed that some patients with Opitz G/BBB syndrome (OMIM]145410) or Cayler cardiofacial syndrome (OMIM]125520) have del.22q11,11,12 and thatthe clinical spectrum associated with del.22q11 is muchwider than was previously recognized as ‘‘CATCH’’.
‘‘22q11DS’’ is the appropriate term used now to refer tothe wide clinical spectrum due to del.22q11.
Clinical Practice for 22q11.2 Deletion Syndrome
Clinical features
The clinical ﬁndings associated with del.22q11 areextensive and highly variable from patient to patient.
Main features include CHD, characteristic facial appearance, immunodeﬁciency from thymic hypoplasia,
velopharyngeal dysfunction with or without cleft palate, hypocalcemia due to hypoparathyroidism, developmental and behavioral problems, and psychiatricdisorders in adulthood. Over 180 minor manifestations
have been reported in association with 22q11DS, suchas renal anomalies, short stature, anal atresia, inguinal
hernia, hypospadias, squint, facial nerve palsy, scoliosis and thrombocytopenia [http://www.vcfsef.org/
facts.html].
CHD: Approximately 75% of patients with 22q11
DS have CHD.13 The types of CHD are characterized
as conotruncal and aortic arch defects including tetralogy of Fallot (TOF) (@30%), interrupted aortic arch
(IAA) type B (@15%), ventricular septal defect (VSD)(@15%), persistent truncus arteriosus (PTA) (@10%)
and others (@5%). Del.22q11 is the second most common genetic cause of CHD and is present in @60% of
patients with IAA type B, @35% of patients with PTA
and @15% of patients with TOF (@55% of patientswith TOF plus pulmonary atresia and major aortopulmonary collateral arteries).14,15 CHD signiﬁcantly
affects the prognosis of patients with 22q11DS and is
the main cause of mortality. Full evaluations, surgicalplans and life-time follow-up for CHD may be required.
Characteristic facial appearance: Almost all patients
have some of the following facial features: lateral displacement of inner canthi, narrow palpebral ﬁssures,
bloated eyelids, small mouth, hypoplastic mandible,
deformed ear lobe, ﬂat nasal bridge and square nosedue to hypoplastic ala nasi (Fig. 1).15,16 The facial
appearance is most pronounced during childhood, andFig. 1 A two year-old boy with 22q11.2 deletion syndrome who has
characteristic facial appearance and tetralogy of Fallot (left), and theauthor.

is sometimes not apparent in neonates or adults. Although it is relatively easy to diagnose patients withtypical facial appearance, some patients only have apart of the characteristic facial features making the diagnosis difﬁcult.
Immunodeﬁciency from thymic hypoplasia: Despite
the emphasis on thymus involvement in DGS,5 a severe
T cell immunodeﬁciency, which may require immune
reconstruction by thymus or bone marrow transplantation during early infancy, is present only in less than 5%of patients with 22q11DS.13 Maldevelopment of thethymus, however, is common, leading to an absence ofmediastinal thymic tissue in over 50% of the patients.
Diminished T cell counts in the peripheral blood andslow declines in T cell population with aging are observed in these patients.17 In patients with 22q11DS,
recurrent infections such as bronchopneumonia andotitis media are also common, but have little relationship to speciﬁc immunologic laboratory features. These
infections may be associated with false aspiration due tovelopharyngeal dysfunction and/or structural anomaliesof the inner ear (described below). Most patients with
22q11DS can safely be immunized and generate sufﬁcient antibody responses.18
Velopharyngeal dysfunction with or without cleft palate: Velopharyngeal dysfunction can occur for a varietyof reasons. In case of structural abnormalities, the termvelopharyngeal insufﬁciency is more appropriate. Cleft
palate is present in @10% of patients with 22q11DS.13
An open cleft is easily recognized, but it requires careful examination to diagnose a submucous cleft. Functional velopharyngeal inadequacy, the more appropriate term for which is velopharyngeal incompetence, ispresent in more than 30% of patients with 22q11DS

Keio J Med 2002; 51 (2): 77–88

with a wide spectrum of severity.13,19,20 Poor mobilityof the soft palate can result in the patients’ inability toclose the velopharyngeal aperture.
The velopharyngeal dysfunction leads to several agedependent problems.19 In early infancy, it leads to anincreased frequency of feeding problem, which often
need the placement of a temporary feeding tube, mainly as a result of a poor sucking reﬂex, a failure of generation of negative pressure suction in oral cavity, andfrequent nasal regurgitation. It may also lead to anincreased frequency of aspiration pneumonia. In older
children, velopharyngeal dysfunction can result in hypernasality, nasal escape creating a poor speech intelligibility and subsequent compensatory speech related
behavior.
When evaluating velopharyngeal function, the patient has to repeat standard words and sentences, so
cooperation of the patient is essential. Because ofdevelopmental delay and behavioral problems, these
diagnostic procedures are often difﬁcult to perform inpatients under the age of 5 years with 22q11DS. Direct
assessment of the velopharyngeal function by viewing
the velopharyngeal isthmus and pharynx through combined nasopharyngoscopy and multi-view radiologicalimaging is possible.21–23
For patients with mild hypernasality, speech therapy
should be started as early as possible to improve velar
function by using nasal versus oral airstream exercises
and palatal exercises like blowing. The speech therapist
must be aware of the learning disabilities and concentration problems in patients with 22q11DS that may
negatively inﬂuence the outcome of the speech therapy.
A pharyngoplasty is recommended in cases of velopharyngeal dysfunction with moderate or severe hypernasal resonance and/or nasal escape.24 This severe
dysfunction should be corrected as early as possible, but
may be delayed until after surgery for CHD in patientswith 22q11DS.
In patients with 22q11DS, recurrent otitis media andconductive hearing loss as a result of chronic serous
otitis media are common.13,20 Although velopharyngeal
dysfunction and/or structural abnormalities of inner ear
may play a role in these conditions, speciﬁc and systematic knowledge regarding the ear diseases in 22q11DS
is still scarce.
Hypocalcemia due to hypoparathyroidism: Approximately 70% of patients of 22q11DS manifest variable
degrees of hypocalcemia reﬂecting low serum parathyroid hormone (PTH).13,25 In the severe cases, hypocalcemia is congenital, with clinical manifestations
requiring immediate treatment and continuous administration of active vitamin D and/or calcium.
In patients with 22q11DS, latent hypoparathyroidism
is more common than persistent symptomatic hypo-79

calcemia.26,27 While most patients have transient neonatal hypocalcemia with spontaneous resolution in theﬁrst years of life, recurrence may be observed later inchildhood or adolescence. In patients with latent hypoparathyroidism, the parathyroid secretion is sufﬁcientunder basal conditions, but cannot increase in response
to hypocalcemic stress, thus leading to hypocalcemia.
Such hypocalcemic stress may result from inadequate
intakes of calcium, especially when the calcium needs
are highest because of rapid skeletal growth, like during
infancy, adolescence or pregnancy.27–29 Normal valuesof serum calcium and PTH are found in basal condition. But a reduced PTH response to hypocalcemic
stress, reﬂecting partial hypoparathyroidism, may beunmasked by ethylenediaminetetraacetic acid (EDTA)
infusions.30,31 In cases where the ﬁrst clinical sign is
generalized convulsion due to late onset hypocalcemia,
the patient is often misdiagnosed as having epilepsy.
These observations emphasize the need for a regularand continuous survey of the parathyroid status inpatients with 22q11DS. Factors or events linked to theother features of the 22q11DS should be considered asthey may induce transition from latent to overt hypocalcemia. For example, for surgery of CHD or cleft
palate, patients may require calcium supplements while
oral intakes are limited. During the treatment of hypocalcemia in patients with 22q11DS, serum calcium
should be controlled at low normal levels, above thelevels inducing clinical manifestations of hypocalcemia
and below those inducing hypercalciuria, which may
result in renal stones since the reabsorption of calciumin the kidney is reduced because of low PTH.
Developmental and behavioral problems: Most
patients with 22q11DS have cognitive deﬁcits ranging
from learning disabilities to mental retardation.13 The
cognitive deﬁcits are not dependent on the presenceof associated anomalies.32,33 Mean intelligence quality(IQ) is about 75, but severe mental retardation israre.33,34 Slow language development and delayeddevelopmental milestones are common in early infancy.
Learning disabilities, better verbal abilities than performal abilities, and poor attention and concentration
are characteristic. Although most patients can go toregular school, preschool evaluation and early educational intervention for each individual are recommended. The intervention should include training ofboth verbal (language, articulation, reading, comprehension) and nonverbal areas (motor skills, visualspatial organization, general mathematical training, attention, and social skills), and speech therapy (see thesection of velopharyngeal dysfunction) if necessary.
Psychiatric disorders in adulthood: Recent studies
have reported an increased incidence (@20%) of psy-

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Yamagishi H: The 22q11.2 deletion syndrome

Fig. 2 Genes and deletions in human chromosome 22q11 region and its syntenic region of mouse chromosome 16. Upper lines (a-l) show
deleted regions of human patients. a, 3 Mb typically deleted region (TDR); b, smaller 1.5 Mb deletion; c-l, atypical deletions reported in 10
individuals (Ref. 37–44). Lower lines (m-s) show deleted regions of mouse chromosome 16 deletion mutant models. Note that Tbx1 is located in
the region responsible for aortic arch defects (AA region) which is the shortest overlapping region of (m), (p), (r) and (s), excluding (n). Gene
names: DGCR6, DiGeorge critical region gene 6; PRODH, proline dehydrogenase; IDD, integral membrane protein deleted in DGS; TSK1/2,
threonine/serine kinase-1/2; ES2, expressed sequence-2; GSCL, goosecoid-like; CTP, citrate transport protein; HIRA, histone regulatory A;
NLVCF, nuclear localization signal protein in VCFS; UFD1L, ubiquitin fusion degradation 1-like; CDC45L, cell division cycle 45-like; TMVCF,
transmembrane protein in VCFS; CDCrel1, cell division cycle-related-1; GP1BB, glycoprotein-1bb; TBX1, T-box protein-1; GNB1L, guaninenucleotide-binding protein b-1-like; TRXR2, thioredoxin reductase-2; COMT, catechol-O-methyltransferase; ARVCF, armadillo repeat gene in
VCFS; T10, transcript-10; RANBP1, Ran-binding protein-1; ZNF74, zinc-ﬁnger protein 74; CRKL, v-crk virus oncogene-like; LZTR1, leucinezipper transcriptional regulator-1.

chiatric illness, including schizophrenia and bipolar
disorder, in adolescents and adults with 22q11DS.13
Several initial studies indicate that the rate of del.22q11
in schizophrenia is estimated at 2–5% which may behigher in subpopulations with developmental delay.35,36
Comprehensive studies of adults with 22q11DS are ongoing to further delineate the phenotype and natural
history of associated psychiatric disorders.
Diagnosis and Management
In patients with 22q11DS, the remarkable variability
in clinical expression remains a major limiting factor inearly diagnosis, and represents a challenge to management and follow-up. To diagnose patients with partial
syndrome or atypical manifestations requires careful
clinical observation. Conotruncal and aortic arch defects
described above are the most early and major signs that
help the diagnosis. Minor manifestations almost always
are associated, such as minor facial anomalies, signs ofvelopharyngeal dysfunction, and slender appearance of

the ﬁngers. These ﬁndings are non-speciﬁc, but theirabsence makes the diagnosis of 22q11DS unlikely. A
major anomaly (CHD, cleft palate, symptomatic hypocalcemia) should alert the clinician to look for minor
manifestations of the syndrome. Prominent features
helpful for diagnosis are variable depending on thepatients’ age: CHD, severe immunodeﬁciency (rare) andsymptomatic hypocalcemia during early infancy; velopharyngeal dysfunction and developmental delay inpreschool age; learning disabilities, latent hypoparathyroidism and psychiatric disorders from school age
to adulthood.
A routine test based on ﬂuorescence in situ hybridization (FISH) using probes (TUPLE1 or N25) fromthe critical deletion region in 22q11.2 is available inmost cytogenetic laboratories to conﬁrm diagnosis
for patients with both typical and atypical clinical presentations. Rarely, deletions are present in 22q11.2,
but not detectable by routine FISH analysis. About 10
cases of such atypical deletions of 22q11.2 have beenreported (Fig. 2).37–44 Del.22q11 is not detectable byKeio J Med 2002; 51 (2): 77–88
Table 1

81

Management Program for Patients with 22q11.2 Deletion Syndrome at Keio University Hospital
Newborn & Infant

Velopharyngeal
dysfunction

Assessment of velopharyngeal function
Continuous speech therapy
and ear problem
Plastic surgery (if necessary)
Pharyngoplasty (if necessary)and speech therapy
Assessment of speech (pre-school)
Periodic serum calcium exam
Evaluation of latenthypocalcemia
Oral administration of active vitamin D (if necessary) ! urinary calcium (and renal
ultrasound) check
Calcium supplement at perioperative
periods
Assessment of developmental quality
Assessment of intelligence
quality (pre-school)
Intervention for developmental delay
Intervention for learning
disabilities
Psychiatrist
consultation
Regular measurements of body size
Hormonal evaluation
Screening renal ultrasound
Teeth care (Dentist consultation)
Pediatric surgery (anal atresia, inguinal hernia, etc.)
Ophthalmology (squint, etc.)
Orthopedics (scoliosis, talipes
equinovarus, etc)

Developmental delay

Psychiatric disorder
Short stature
OthersVaccination

extensive FISH analysis in approximately 10% of
patients who are clinically diagnosed as having DGS or
22q11DS. These patients include those who have chromosome deletion of 10p or others,45,46 and those whohave undeﬁned genetic etiology.
Comprehensive evaluation and management ofpatients with 22q11DS are required for multisystemdisorders. The primary care physician (pediatrician inmost cases) has an important role in caring for thepatients and their families, and needs to collaborate
with many specialists of the associated abnormalities.
Table 1 shows ‘‘Management program for patients with
22q11DS’’ in Department of Pediatrics of Keio University Hospital.
Clinical and Basic Research for 22q11.2 Deletion
Syndrome
Molecular genetics
Despite the heterogeneous clinical presentations,remarkably homogenous deletions in 22q11.2 region
are present in patients with 22q11DS, and only a small
number of atypical deletions have been reported. Approximately 90% of patients have a typical deleted re-Regular follow-up

Puberty

Palliative operation

Blood exam (Immunodeﬁciency
screening)
Immune reconstruction (severe cases)
Otolaryngeal exam

Corrective operation

School-age

Congenital heartdiseases
Immunodeﬁciency

Hypocalcemia

Regular follow-up

Toddler

Restriction in activity (ifnecessary)
Vaccination

gion (TDR) of @3 Mb, which encompasses an estimated 30 genes, whereas about 8% of patients have asmaller deletion of @1.5 Mb, which encompasses 24
genes (Fig. 2; Reviewed in 47). Recent molecularstudies characterized low copy repeat sequences in the
22q11 region (LCR22), which ﬂank the TDR and the
1.5 Mb deletion region. Unequal recombination events
between ﬂanking LCRs explain the recurrence of deletions of uniform size.48 A similar mechanism of action
has been described for other chromosomal deletion
syndromes, such as Williams syndrome, and PraderWilli and Angelman syndrome.49–51
The basis of phenotype variability of 22q11DS
remains to be elucidated. There is no genotypephenotype correlation.52–55 There is phenotype variability even in familial cases, including individuals withmonozygotic twins, although affected family members
have presumably inherited identical deletions.16,56,57
Suggested explanations for the variable phenotype include allelic variability, variable penetrance and variable expressivity caused by environmental factors or
stochastic events during fetal development. As a matterof fact, exposure of early human and mouse embryos
to high concentration of vitamin A produces a phenocopy of DGS, and some patients with fetal alcohol syn-82

drome have some phenotype overlapping with that of
22q11DS.58
Developmental biology
The structures primarily affected in patients with
22q11DS are derivatives of the embryonic pharyngeal
arches and pouches, suggesting that 22q11DS is a
developmental ﬁeld defect of the pharyngeal apparatus.
The pharyngeal arches are bilaterally symmetric structures that develop in a segmental fashion along the anterior-posterior axis, and are composed of a number ofdifferent embryonic cell types (Fig. 3).59 Each arch has
an outer covering of ectoderm, an inner covering of
endoderm and a central core of paraxial mesoderm adjacent to an pharyngeal arch artery. Cephalic neuralcrest cells migrate from the neural fold in the caudal
hindbrain to each pharyngeal arch, where they form themesenchyme that surrounds the pharyngeal arch arteries and the mesodermal core. The mesoderm gives rise
to facial and neck muscles, while the neural crestderived mesenchyme contributes to skeletal structuresof the face as well as cardiovascular structures. Pharyngeal pouches, formed by the endoderm-derived epithelial layer, give rise to craniofacial organs, including thethymus and parathyroid glands, whereas pharyngeal
clefts, formed by the ectoderm-derived epithelial layer,
give rise to the epidermis and the sensory neurons of

Yamagishi H: The 22q11.2 deletion syndrome

the arch associated ganglia.
Experiments on chick embryos involving ablation of
cephalic neural crest cells before their migration fromthe neural fold result in a phenocopy of the main features of 22q11DS.60,61 Mutations of genes implicated inneural crest development in mouse, such as Pax3 andendothelin-1, can similarly give malformations reminiscent of 22q11DS.62,63 Based on these observations,
22q11DS has been believed to result primarily fromabnormal development of neural crest cells that populate the pharyngeal arches, their corresponding pharyngeal arch arteries, and conotruncal region of theheart.
Candidate genes
Approach using experimental animals: Although extensive gene searches have been successful in identifying more than 30 genes in the TDR of 22q11.2 (Fig. 2),
standard analyses so far have failed to detect mutations in any of these genes in patients with DGS or
VCFS phenotype but lacking a cytogenetic aberration(Reviewed in 64). From the aspects of molecular genetics and developmental biology of 22q11DS described above, the gene(s) responsible for phenotype of
22q11DS should be haploinsufﬁcient gene(s) within
22q11.2 region that are expressed and play an important role in developing pharyngeal arches.65

Fig. 3 Development of pharyngeal arch structures. (A) Right lateral view of a mouse embryo at embryonic day (E) 9.5 (equivalent to about 28
days in a human embryo). Arrows show the migration of cephalic neural crest cells derived from the dorsal aspect of neural tube (neural fold)into the head, pharyngeal arches and the conotruncal region (outﬂow tract) of the heart. (B) Coronal section of pharyngeal arches corresponding to E9.5–10.5 mouse or stage 16–18 chick embryos. Each pharyngeal arch is composed of an outer epithelium of ectoderm (black),
central core of paraxial mesoderm (blue) adjacent to a pharyngeal arch artery, and an inner epithelium of endoderm (green) which forms
pharyngeal pouches between each pharyngeal arch. Neural crest cells migrate from the caudal hindbrain and form the mesenchyme (red) thatsurrounds the pharyngeal arch arteries and the mesodermal core. Arabic numerals represent 1st to 4th pharyngeal arches, and Roman numerals
represent 1st to 4th pharyngeal pouches. Note that 1st and 2nd pharyngeal arch defects lead to minor facial anomalies and cleft palate, 3rd and4th pouch defects lead to hypoplasia of thymus and parathyroid glands, 3rd and 4th arch artery defects lead to aortic arch defects, and defects ofthe conotruncal region lead to congenital heart defects such as tetralogy of Fallot and persistent truncus arteriosus.

Keio J Med 2002; 51 (2): 77–88

The use of experimental animal models has suggested several attractive candidate genes. HIRA encodesa mammalian ortholog of the yeast Hir1p and Hir2p
proteins, which are corepressors of cell cycle-dependent
histone gene regulation, and are expressed in neuralcrest-derived tissues as well as some other tissues inboth chick and mouse embryos.66–68 Embryonic attenuation of Hira by antisense oligonucleotides in chickcardiac neural crest cells results in an increased incidence of PTA.69 Hira interacts biochemically with Pax3
which is a homeodomain protein implicated in neuralcrest development.70
UFD1L, the ortholog of a highly conserved yeast
gene involved in ubiquitin-mediated protein degradation, is also expressed strongly in the pharyngeal arches,head region and limb buds in both chick and mouseembryos.38,71 Ufd1l was identiﬁed as a downstream
gene of a bHLH transcription factor, dHAND, and was
downregulated in dHAND-null mouse embryos that
display pharyngeal arch hypoplasia and cardiac outﬂow
tract defects.38 Antisense attenuation of Ufd1l in chickcardiac neural crest cells leads to increased incidence of
septation failure of cardiac outﬂow tract (unpublishedobservation, 2001). These observations suggest that
HIRA and UFD1L may contribute to some aspects ofthe 22q11DS phenotype. However, mice heterozygous
for either Hira or Ufd1l mutation are apparently normal
and homozygotes die during early embryogenesis.72,73
Crkol (CRKL in human) encodes an SH2-SH3-SH3
adapter protein involved in growth-factor response andfocal-adhesion signaling, and is expressed in tissues
derived from neural crest cells during development.74
Mice homozygous null for Crkol are embryonic lethal,and develop a similar phenotype to that of 22q11DS,
including conotruncal and aortic arch defects, thymus
and craniofacial abnormalities, suggesting a potential
contribution of CRKL to the 22q11DS phenotype.74
However, mice heterozygous for Crkol mutation arenormal and viable, indicating that Crkol is not haploinsufﬁcient, at least in the mouse.
COMT, which encodes catecol-O-methyltransferase
and PRODH, which encodes proline dehydrogenase,are potentially involved in the behavioral and psychiatric phenotype of 22q11DS.75–77 Comt mutant mice display impairment in emotional behavior. Prodh mutant
mice have a deﬁcit in sensorimotor gating that is aneural ﬁltering process, that allows attention to be focused on a given stimulus, and is affected in patientswith some psychiatric disorders.
Approach using genomic engineering in the mouse:
Recent advanced genome manipulation technologies inmice have more clearly provided an insight into the roleof candidate genes for 22q11DS.47 The human region
on chromosome 22q11 and the syntenic, or orthologous,

83

region in the mouse on chromosome 16 (MMU16) arevery similar in gene content, although they have a different genomic organization. Several groups have generated deletions in mice in the region on MMU16 toextend the human molecular genetic approaches to
identify genes responsible for 22q11DS (Fig. 2). Lindsay
et al. ﬁrst generated mice with a hemizygous deletionfrom Es2 to Ufd1l, named Df1/þ, which are carrying
one copy of 18 mouse homologues out of the 24 genesthat are deleted in patients with the 1.5 Mb deletion.73
Df1/þ mice exhibited aortic arch defects in about 30%
of cases at initial analyses. Mice with a homozygous
deletion of Df1 region showed early embryonic lethality. Aortic arch anomalies could be corrected in micethat were bred to carry the Df1 deletion on one chromosome 16 and a reciprocal duplication on the other,which restored normal gene dosage. This genetic complementation examination suggested that the aorticarch anomalies observed in Df1/þ mice were due tohaploinsufﬁciency of a gene(s) mapped within the Df1
region. Mice with a hemizygous deletion of @150 kb of
the proximal region of the TDR, which encompasses 7
genes, and mice with a hemizygous deletion of a 550 kb
region, which encompasses 16 genes partially overlapping Df1 were subsequently generated by Kimber et
al. and Puech et al., respectively, but neither of themshowed any phenotype of 22q11DS.78,79 Later, micewith hemizygous deletion from Idd to Hira (Lgdel/þ), a
region containing the Df1/þ region plus 5 more genes,
generated by Merscher et al., exhibited similar types ofaortic arch anomalies as Df1/þ in about 50% of cases,
as well as parathyroid gland aplasia.80 Lindsay et al.
and Merscher et al. independently narrowed the critical region using chromosomal engineering and rescue
transgenesis, and came to the same conclusion that Tbx1
is responsible for the aortic arch phenotype observed in
Df1/þ and Lgdel/þ mice. Further establishment andanalysis of mice heterozygous for Tbx1 mutation alone
by both groups supported this conclusion.72,80
TBX1: TBX1 belongs to the T-box family of transcription factors.81 Members of this family are involvedin early development of both vertebrates and invertebrates, including speciﬁcation of mesoderm and endoderm, and heart and limb morphogenesis (Reviewed in82). Jerome and Pappaioannou independently reported
mice homozygous and heterozygous for a Tbx1 mutation allele.83 According to their report, mice homozygous for Tbx1 mutation died perinatally and displayed
virtually all testable features of 22q11DS, including
conotruncal defects, abnormal facial features, cleft
palate, and hypoplasia of the thymus and parathyroid
glands. Mice heterozygous for Tbx1 mutation have only
aortic arch patterning defects with a less penetrant
phenotype, although they do not reﬂect the entire phe-

84

Yamagishi H: The 22q11.2 deletion syndrome

Fig. 4 Expression patterns of Tbx1. Mouse (A–C) and chick (D–F) embryos and primary neural crest culture from chick embryos (G–I) are
shown. RNA in situ hybridizations for whole-mount (A, D), coronal section (B, C) and transverse section (E, F) demonstrate Tbx1 expression(blue or white signals) in the mesodermal core (arrows) and endodermal epithelium (arrow heads) of pharyngeal arches, head mesenchyme(hm) and otic vesicle (ov). B and E are bright ﬁeld images of C and F, respectively. Asterisks indicate pharyngeal arch arteries. In chick primaryneural crest culture cells, HNK-1 (G) and neuropilin-1 (H) are detected by immunocytochemistry and mRNA in situ hybridization, respectively,
but Tbx1 is not detectable by mRNA in situ hybridization (I). fg, forgut; da, dorsal aorta; ht, heart.

notypic spectrum of 22q11DS. These results provided
evidence for TBX1 likely being a major genetic determinant in the etiology of 22q11DS. Importantly, miceheterozygous for Tbx1 showed a varying degree ofabsent or reduced 4th pharyngeal arch arteries, later
resulting in aortic arch anomalies, depending on their
genetic background. This phenotype nicely matches
the abnormalities observed in Df1/þ and Lgdel/þ
mice, consistent with the analyses of the two othergroups.72,80,83 However, mutational analysis of thecoding region of TBX1 in over 200 patients with a clinical suspicion of 22q11DS but without deletion or otherdetectable rearrangements has so far failed to identify
TBX1 gene mutations.72,84
To determine the relevance of Tbx1 to the pathogenesis of 22q11DS, we performed detailed expression
analyses of mouse and chicken Tbx1 in pharyngeal archdevelopment (Fig. 4).85 In mouse embryos at embryonic day (E) 9.5, Tbx1 was preferentially expressed in
the pharyngeal arches, the ventral half of the otic vesicle, and in the head. Section in situ hybridization at
E10.5 showed expression of Tbx1 in the mesodermalcore, adjacent to the pharyngeal arch arteries, and theendodermal epithelium of pharyngeal arches as wellas in the head mesenchyme. Whole mount and section
in situ hybridization using chicken Tbx1 riboprobedemonstrated a similar expression pattern to that ofmouse Tbx1. Interestingly, Tbx1 was not detectable inneural crest-derived mesenchyme in the pharyngeal
arches of both mouse and chicken embryos. The expression analysis of Tbx1 in the primary chicken neuralcrest cells further clearly demonstrated that Tbx1 was
not expressed in neural crest cells.
Future directions
A ﬁnding that TBX1 haploinsufﬁciency is likely to be
the major determinant of aortic arch defects in patientswith 22q11DS is a signiﬁcant advancement in this ﬁeld.
The pathogenetic basis of 22q11DS would become
more clear by future efforts to identify the genes thatare targets of TBX1, to deﬁne molecular pathways
controlling TBX1 expression, and to elucidate the roleof TBX1 in cardiovascular and pharyngeal arch development. Although functional mutations have notbeen identiﬁed in patients with a clinical suspicion of

Keio J Med 2002; 51 (2): 77–88

22q11DS but without the deletion, further extensive
mutational analyses are indicated for coding, non coding and regulatory regions of TBX1.
The delineation of the expression pattern of Tbx1
sheds light on the molecular and cellular basis of
22q11DS. Interestingly, Tbx1 is strongly expressed in
endoderm and mesoderm cells, but not in neural crest
cells during the pharyngeal arch development.81,83,85
This result suggests that the primary lesion of the mainfeatures in 22q11DS may not be the neural crest cells,
although 22q11DS has been believed to result primarily
from abnormal development of neural crest cells as
mentioned above. Numerous recent studies have demonstrated that appropriate epithelial-mesenchymal signaling is essential for proper development of the pharyngeal arches, and that the primary impairment of theepithelial endoderm can secondarily affect migration ordifferentiation of neural crest cells during the pharyngeal arch development.86–88 Importantly, mutations
in genes expressed in either neural crest cells or nonneural crest cells in pharyngeal arches can result insimilar aortic arch defects in mice. Future studies arethus required to focus on the signals that mediate interactions between neural crest cells and endoderm cells
and/or mesoderm cells in pharyngeal arches to uncover
the developmental mechanism of 22q11DS.
Although Tbx1 haploinsufﬁciency accounts for theaortic arch defects in Df1/þ mice, haploinsufﬁciency of
TBX1 may not be the only cause of the entire clinicalpresentations of 22q11DS. Df1/þ mice have broader
phenotypic features that have not been described inmice heterozygous for Tbx1 mutation, including conotruncal defects (overriding aorta plus ventricularseptal defect), hypoplasia of thymus and parathyroid
glands, and the behavioral phenotype, still Df1/þ mice
do not cover the entire phenotypic spectrum of
22q11DS.73,89,90 The contribution of modiﬁer genes
and of additional genes either within (e.g. Ufd1l, Comtand Prodh) or outside (e.g. Hira and Crkol ) of Df1 region on 22q11.2, and elsewhere to various features of
22q11DS, as well as the genetic interaction between
these genes and Tbx1, remain to be studied.
TBX1 haploinsufﬁciency also cannot account forthe several reported patients with 22q11DS who havedeletions excluding TBX1 locus. The presence of longrange cis-acting genetic elements that regulate theresponsible gene(s) on 22q11.2 has been proposed to
explain the similar phenotype of patients with atypicaldeletions.37 In Df1/þ mice, however, a region of only
140 kb, containing Tbx1 and 3 more genes, can fully
rescue the aortic arch phenotype when it is introduced
using transgenic mice, thus excluding the existence oflong-range cis-acting elements regulating Tbx1, at leastin the mouse model.72 It is unknown whether suchgenetic elements may be present in humans, but not85

conserved in mice.
Finally, the research advances described here should
be beneﬁcial to patients with 22q11DS. The continuous
efforts to delineate the clinical features with accurate
prevalence will lead to effective evaluation and intervention for the patients. Better knowledge for the behavioral and psychiatric phenotype and the phenotype
in adult age need to be accumulated over the next
few years. One of the ultimate goals for basic research
is preventive treatment (and eventual gene therapy)
of afﬂicted infants after discovery of the responsible
gene(s) and their function, although it requires much
more progress to achieve these interventions. Searchingfor the upstream or downstream targets of Tbx1 may
be the next key step toward the goal. A multifaceted
approach to dissect the clinical and genetic aspects of
22q11DS would develop a deeper understanding anda better intervention for patients with 22q11DS or thevariety of disorders associated with this syndrome.
Acknowledgments: The author wishes to thank the patient and his
family for the photograph (the permission from the family has beenobtained, and the permission for the use of the photograph in thispublication has been obtained from the family). The author also
wishes to thank Dr. Deepak Srivastava for critical reading of themanuscript, Dr. Chihiro Yamagishi for preparation of the ﬁgures, andDr. Yoshifumi Kojima and members of Division of Pediatric Cardiology in Keio University School of Medicine, and Prof. Nobutake
Matsuo for their support. Expression analyses of Tbx1 was performedin Dr. Srivastava’s laboratory at University of Texas Southwestern
Medical Center.

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