REVIEW The 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

(Receivedfor publicationon March27, 2002)

Abstract. The 22q11.2 deletion syndrome (22q11DS) encompasses DiGeorge syndrome, velo-cardio facial 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 is necessary for patients with 22q11DS. A striking aspect of the 22q11DS phenotype is its variability, the basis 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 and pouches suggesting that haploinsufficiency of the gene(s) on the deleted region, spanning 2-3 Mb, is important 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 the developmental role of some of these genes, including TBX1. In this review, the clinical features and management of patients with 22q11DS are integrated with our current understanding of the embryo logical 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, hypocal cemia, pharyngeal arch development

Introduction ism and immune deficiency in 1965;5 VCFS was asso ciated with cleft palate, CHD, a distinct facial appear The entity of 22q11.2 deletion syndrome ance, and learning difficulties in 1978;6 CAFS was described in 1976 and was characterized by conotruncal A heterozygous deletion within chromosome 22q11.2 CHD, a distinct facial appearance and hyper-nasal (del.22q11) is the genetic basis of the most common voice.7 Clinical genetics have gained from the discovery interstitial deletion syndrome, the 22q11.2 deletion that these syndromes have overlapping clinical pre syndrome (22q11DS) in humans, with an incidence of sentations and share a common chromosomal deletion. 1 in 4000-5000 births.1 Most are sporadic in origin, DGS, VCFS and CAFS are, therefore , part of 22q11DS but 10-20% of deletions are inherited as an autosomal reflecting various outcomes of the same underlying ge dominant trait. In the early 1990's, the deletion was netic defect. reported in association with three distinct syndromes, The acronym "CATCH22 (cardiac defects, abnormal namely DiGeorge syndrome (DGS; OMIM#188400), facies, thymic hypoplasia, cleft palate, hypocalcemia , velo-cardio-facial syndrome (VCFS; OMIM#192430) and 22q11 deletions)" was proposed, in 1993, to aid and conotruncal anomaly face syndrome (CAFS; in remembering the main features of the syndrome OMIM#217095).2-4 DGS was originally characterized encompassing DGS, VCFS and CAFS.8 However , clini by congenital heart defects (CHD), hypoparathyroid cal use of this term may be inappropriate because of

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: [email protected]

77 78 Yamagishi H: The 22q11.2 deletion syndrome following reasons:9 1) The term "Catch-22" has a nega tive meaning which represents a situation where it is impossible for you to do anything, originally from Hel ler's novel entitled "CATCH 22";10 2) The term "A" referring to "abnormal facies" is difficult to accept for patients and their family; 3) Recent studies have revealed that some patients with Opitz G/BBB syn drome (OMIM#145410) or Cayler cardiofacial syn drome (OMIM#125520) have del.22q11,11.12and that the clinical spectrum associated with del.22q11 is much wider than was previously recognized as "CATCH". "22q11DS" is the appropriate term used now to refer to the wide clinical spectrum due to del.22q11.

Clinical Practice for 22q11.2 Deletion Syndrome Fig. 1 A two year-old boy with 22q11.2 deletion syndrome who has characteristic facial appearance and tetralogy of Fallot (left), and the Clinical features author.

The clinical findings associated with del.22q11 are extensive and highly variable from patient to patient. is sometimes not apparent in neonates or adults. Al

Main features include CHD, characteristic facial ap though it is relatively easy to diagnose patients with pearance, immunodeficiency from thymic hypoplasia, typical facial appearance, some patients only have a velopharyngeal dysfunction with or without cleft pal part of the characteristic facial features making the di ate, hypocalcemia due to hypoparathyroidism, devel agnosis difficult. opmental and behavioral problems, and psychiatric disorders in adulthood. Over 180 minor manifestations Immunodeficiency from thymic hypoplasia: Despite have been reported in association with 22q11DS, such the emphasis on thymus involvement in DGS,5 a severe as renal anomalies, short stature, anal atresia, inguinal T cell immunodeficiency, which may require immune hernia, hypospadias, squint, facial nerve palsy, sco reconstruction by thymus or bone marrow transplanta liosis and thrombocytopenia [http://www.vcfsef.org/ tion during early infancy, is present only in less than 5% facts.html]. of patients with 22q11DS.13 Maldevelopment of the thymus, however, is common, leading to an absence of

CHD: Approximately 75% of patients with 22q11 mediastinal thymic tissue in over 50% of the patients. DS have CHD.13 The types of CHD are characterized Diminished T cell counts in the peripheral blood and as conotruncal and aortic arch defects including tetral slow declines in T cell population with aging are ob ogy of Fallot (TOF) (•`30%), interrupted aortic arch served in these patients.17 In patients with 22q11DS,

(IAA) type B (•`15%), ventricular septal defect (VSD) recurrent infections such as bronchopneumonia and (•`15%), persistent truncus arteriosus (PTA) (•`10%) otitis media are also common, but have little relation and others (•`5%). Del.22q11 is the second most com ship to specific immunologic laboratory features. These mon genetic cause of CHD and is present in •`60% of infections may be associated with false aspiration due to patients with IAA type B, •`35% of patients with PTA velopharyngeal dysfunction and/or structural anomalies and •`15% of patients with TOF (•`55% of patients of the inner ear (described below). Most patients with with TOF plus pulmonary atresia and major aorto 22q11DS can safely be immunized and generate suffi pulmonary collateral arteries).14,15 CHD significantly cient antibody responses.18 affects the prognosis of patients with 22q11DS and is the main cause of mortality. Full evaluations, surgical Velopharyngeal dysfunction with or without cleft pal plans and life-time follow-up for CHD may be required. ate: Velopharyngeal dysfunction can occur for a variety of reasons. In case of structural abnormalities , the term Characteristic facial appearance: Almost all patients velopharyngeal insufficiency is more appropriate . Cleft have some of the following facial features: lateral dis palate is present in •`10% of patients with 22q11DS.13 placement of inner canthi, narrow palpebral fissures, An open cleft is easily recognized, but it requires care bloated eyelids, small mouth, hypoplastic mandible, ful examination to diagnose a submucous cleft. Func deformed ear lobe, flat nasal bridge and square nose tional velopharyngeal inadequacy, the more appropri due to hypoplastic ala nasi (Fig. 1).15.16 The facial ate term for which is velopharyngeal incompetence , is appearance is most pronounced during childhood, and present in more than 30% of patients with 22q11DS Keio J Med 2002; 51 (2): 77-88 79

with a wide spectrum of severity.13,19.20 Poor mobility calcemia.26.27 While most patients have transient neo of the soft palate can result in the patients' inability to natal hypocalcemia with spontaneous resolution in the close the velopharyngeal aperture. first years of life, recurrence may be observed later in

The velopharyngeal dyssssfunctionleads to several age childhood or adolescence. In patients with latent hypo dependent problems.19 In early infancy, it leads to an parathyroidism, the parathyroid secretion is sufficient increased frequency of feeding problem, which often under basal conditions, but cannot increase in response need the placement of a temporary feeding tube, main to hypocalcemic stress, thus leading to hypocalcemia. ly as a result of a poor sucking reflex, a failure of gen Such hypocalcemic stress may result from inadequate eration of negative pressure suction in oral cavity, and intakes of calcium, especially when the calcium needs frequent nasal regurgitation. It may also lead to an are highest because of rapid skeletal growth, like during increased frequency of aspiration pneumonia. In older infancy, adolescence or pregnancy.27-29 Normal values children, velopharyngeal dysfunction can result in hy of serum calcium and PTH are found in basal condi pernasality, nasal escape creating a poor speech intel tion. But a reduced PTH response to hypocalcemic ligibility and subsequent compensatory speech related stress, reflecting partial hypoparathyroidism, may be behavior. unmasked by ethylenediaminetetraacetic acid (EDTA) When evaluating velopharyngeal function, the pa infusions.30,31 In cases where the first clinical sign is tient has to repeat standard words and sentences, so generalized convulsion due to late onset hypocalcemia, cooperation of the patient is essential. Because of the patient is often misdiagnosed as having epilepsy. developmental delay and behavioral problems, these These observations emphasize the need for a regular diagnostic procedures are often difficult to perform in and continuous survey of the parathyroid status in

patients under the age of 5 years with 22q11DS. Direct patients with 22q11DS. Factors or events linked to the assessment of the velopharyngeal function by viewing other features of the 22q11DS should be considered as the velopharyngeal isthmus and pharynx through com they may induce transition from latent to overt hypo bined nasopharyngoscopy and multi-view radiological calcemia. For example, for surgery of CHD or cleft

imaging is possible.21-23 palate, patients may require calcium supplements while For patients with mild hypernasality, speech therapy oral intakes are limited. During the treatment of hypo should be started as early as possible to improve velar calcemia in patients with 22q11DS, serum calcium function by using nasal versus oral airstream exercises should be controlled at low normal levels, above the and palatal exercises like blowing. The speech therapist levels inducing clinical manifestations of hypocalcemia must be aware of the learning disabilities and concen and below those inducing hypercalciuria, which may tration problems in patients with 22q11DS that may result in renal stones since the reabsorption of calcium negatively influence the outcome of the speech therapy. in the kidney is reduced because of low PTH. A pharyngoplasty is recommended in cases of velo pharyngeal dysfunction with moderate or severe hyper Developmental and behavioral problems: Most nasal resonance and/or nasal escape.24 This severe patients with 22q11DS have cognitive deficits ranging dysfunction should be corrected as early as possible, but from learning disabilities to mental retardation.13 The may be delayed until after surgery for CHD in patients cognitive deficits are not dependent on the presence with 22q11DS. of associated anomalies.32.33 Mean intelligence quality

In patients with 22q11DS, recurrent otitis media and (IQ) is about 75, but severe mental retardation is conductive hearing loss as a result of chronic serous rare. 31,31 Slow language development and delayed otitis media are common.13.20 Although velopharyngeal developmental milestones are common in early infancy . dysfunction and/or structural abnormalities of inner ear Learning disabilities, better verbal abilities than per may play a role in these conditions, specific and system formal abilities, and poor attention and concentration atic knowledge regarding the ear diseases in 22q11DS are characteristic. Although most patients can go to is still scarce. regular school, preschool evaluation and early educa tional intervention for each individual are recom Hypocalcemia due to hypoparathyroidism: Approxi mended. The intervention should include training of

mately 70% of patients of 22q11DS manifest variable both verbal (language, articulation, reading , compre degrees of hypocalcemia reflecting low serum parathy hension) and nonverbal areas (motor skills , visual spatial organization, general mathematical training roid hormone (PTH).13,25 In the severe cases, hypo , at calcemia is congenital, with clinical manifestations tention, and social skills), and speech therapy (see the

requiring immediate treatment and continuous admin section of velopharyngeal dysfunction) if necessary . istration of active vitamin D and/or calcium. In patients with 22q11DS, latent hypoparathyroidism Psychiatric disorders in adulthood: Recent studies is more common than persistent symptomatic hypo have reported an increased incidence (•`20%) of psy 80 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-1) show deleted regions of human patients. a, 3 Mb typically deleted region (TDR); b, smaller 1.5Mb deletion; c-1, 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 Tbxl 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; UFDIL, ubiquitin fusion degradation 1-like; CDC45L, cell division cycle 45-like; TMVCF,

transmembrane protein in VCFS; CDCrell, cell division cycle-related-1; GPIBB, glycoprotein-1bƒÀ; TBXI, T-box protein-1; GNB1L, guanine nucleotide-binding protein ƒÀ-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, leucine

zipper transcriptional regulator-1.

chiatric illness, including schizophrenia and bipolar the fingers. These findings are non-specific, but their disorder, in adolescents and adults with 22q11DS.13 absence makes the diagnosis of 22q11DS unlikely. A Several initial studies indicate that the rate of del.22q11 major anomaly (CHD, cleft palate, symptomatic hypo in schizophrenia is estimated at 2-5% which may be calcemia) should alert the clinician to look for minor higher in subpopulations with developmental delay.35,36 manifestations of the syndrome. Prominent features Comprehensive studies of adults with 22q11DS are on helpful for diagnosis are variable depending on the going to further delineate the phenotype and natural patients' age: CHD, severe immunodeficiency (rare) and history of associated psychiatric disorders. symptomatic hypocalcemia during early infancy; velo pharyngeal dysfunction and developmental delay in Diagnosis and Management preschool age; learning disabilities, latent hypopar athyroidism and psychiatric disorders from school age In patients with 22q11DS, the remarkable variability to adulthood. in clinical expression remains a major limiting factor in A routine test based on fluorescence in situ hybrid early diagnosis, and represents a challenge to manage ization (FISH) using probes (TUPLE1 or N25) from ment and follow-up. To diagnose patients with partial the critical deletion region in 22q11.2 is available in syndrome or atypical manifestations requires careful most cytogenetic laboratories to confirm diagnosis clinical observation. Conotruncal and aortic arch defects for patients with both typical and atypical clinical pre described above are the most early and major signs that sentations. Rarely, deletions are present in 22q11.2, help the diagnosis. Minor manifestations almost always but not detectable by routine FISH analysis. About 10 are associated, such as minor facial anomalies, signs of cases of such atypical deletions of 22q11.2 have been velopharyngeal dysfunction, and slender appearance of reported (Fig. 2).37-44 Del.22q11 is not detectable by Keio J Med 2002; 51 (2): 77-88 81

Table 1 Management Program for Patients with 22q11.2 Deletion Syndrome at Keio University Hospital

extensive FISH analysis in approximately 10% of gion (TDR) of •`3 Mb, which encompasses an esti patients who are clinically diagnosed as having DGS or mated 30 genes, whereas about 8% of patients have a 22q11DS. These patients include those who have chro smaller deletion of •`1.5Mb, which encompasses 24 mosome deletion of 10p or others,45,46 and those who genes (Fig. 2; Reviewed in 47). Recent molecular have undefined genetic etiology. studies characterized low copy repeat sequences in the Comprehensive evaluation and management of 22q11 region (LCR22), which flank the TDR and the patients with 22q11DS are required for multisystem 1.5 Mb deletion region. Unequal recombination events disorders. The primary care physician (pediatrician in between flanking LCRs explain the recurrence of dele most cases) has an important role in caring for the tions of uniform size.48 A similar mechanism of action patients and their families, and needs to collaborate has been described for other chromosomal deletion with many specialists of the associated abnormalities. syndromes, such as Williams syndrome, and Prader Table 1 shows "Management program for patients with - Willi and Angelman syndrome .49-51 22q11DS" in Department of Pediatrics of Keio Uni The basis of phenotype variability of 22q11DS versity Hospital. remains to be elucidated. There is no genotype

phenotype correlation.52-55 There is phenotype vari Clinical and Basic Research for 22q11.2 Deletion ability even in familial cases , including individuals with Syndrome monozygotic twins, although affected family members

have presumably inherited identical deletions .16,56,57 Molecular genetics Suggested explanations for the variable phenotype in clude allelic variability, variable penetrance and vari Despite the heterogeneous clinical presentations, able expressivity caused by environmental factors or remarkably homogenous deletions in 22q11.2 region stochastic events during fetal development . As a matter are present in patients with 22q11DS, and only a small of fact, exposure of early human and mouse embryos number of atypical deletions have been reported. Ap to high concentration of vitamin A produces a pheno proximately 90% of patients have a typical deleted re copy of DGS, and some patients with fetal alcohol syn 82 Yamagishi H: The 22q11.2 deletion syndrome drome have some phenotype overlapping with that of the arch associated ganglia. 22q11DS.58 Experiments on chick embryos involving ablation of cephalic neural crest cells before their migration from Developmental biology the neural fold result in a phenocopy of the main fea tures of 22q11DS.60,61Mutations of genes implicated in The structures primarily affected in patients with neural crest development in mouse, such as Pax3 and 22q11DS are derivatives of the embryonic pharyngeal endothelin-1, can similarly give malformations reminis arches and pouches, suggesting that 22q11DS is a cent of 22q11DS.62.63 Based on these observations, developmental field defect of the pharyngeal apparatus. 22q11DS has been believed to result primarily from The pharyngeal arches are bilaterally symmetric struc abnormal development of neural crest cells that popu tures that develop in a segmental fashion along the an late the pharyngeal arches, their corresponding pha terior-posterior axis, and are composed of a number of ryngeal arch arteries, and conotruncal region of the different embryonic cell types (Fig. 3).59 Each arch has heart. an outer covering of ectoderm, an inner covering of endoderm and a central core of paraxial mesoderm ad Candidate genes jacent to an pharyngeal arch artery. Cephalic neural crest cells migrate from the neural fold in the caudal Approach using experimental animals: Although ex hindbrain to each pharyngeal arch, where they form the tensive gene searches have been successful in identify mesenchyme that surrounds the pharyngeal arch arter ing more than 30 genes in the TDR of 22q11.2 (Fig. 2), ies and the mesodermal core. The mesoderm gives rise standard analyses so far have failed to detect muta to facial and neck muscles, while the neural crest tions in any of these genes in patients with DGS or derived mesenchyme contributes to skeletal structures VCFS phenotype but lacking a cytogenetic aberration of the face as well as cardiovascular structures. Pharyn (Reviewed in 64). From the aspects of molecular ge geal pouches, formed by the endoderm-derived epithe netics and developmental biology of 22q11DS descri lial layer, give rise to craniofacial organs, including the bed above, the gene(s) responsible for phenotype of thymus and parathyroid glands, whereas pharyngeal 22q11DS should be haploinsufficient gene(s) within clefts, formed by the ectoderm-derived epithelial layer, 22q11.2 region that are expressed and play an impor give rise to the epidermis and the sensory neurons of tant 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 corre sponding 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) that surrounds 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 and 4th pouch defects lead to hypoplasia of thymus and parathyroid glands, 3rd and 4th arch artery defects lead to aortic arch defects, and defects of the conotruncal region lead to congenital heart defects such as tetralogy of Fallot and persistent truncus arteriosus. Keio J Med 2002; 51 (2): 77-88 83

The use of experimental animal models has sug region in the mouse on chromosome 16 (MMU16) are gested several attractive candidate genes. HIRA encodes very similar in gene content, although they have a dif a mammalian ortholog of the yeast Hirlp and Hir2p ferent genomic organization. Several groups have gen proteins, which are corepressors of cell cycle-dependent erated deletions in mice in the region on MMU16 to histone gene regulation, and are expressed in neural extend the human molecular genetic approaches to crest-derived tissuesssas well as some other tissues in identify genes responsible for 22q11DS (Fig. 2). Lindsay both chick and mouse embryos.66-68 Embryonic atten et al. first generated mice with a hemizygous deletion uation of Hira by antisense oligonucleotides in chick from Es2 to Ufdll, named Dfl/+, which are carrying cardiac neural crest cells results in an increased inci one copy of 18 mouse homologues out of the 24 genes dence of PTA .69 Hira interacts biochemically with Pax3 that are deleted in patients with the 1.5Mb deletion.73 which is a homeodomain protein implicated in neural Dfl/+ mice exhibited aortic arch defects in about 30% crest development.70 of cases at initial analyses. Mice with a homozygous UFD1L, the ortholog of a highly conserved yeast deletion of Dfl region showed early embryonic lethal gene involved in ubiquitin-mediated protein degrada ity. Aortic arch anomalies could be corrected in mice tion, is also expressed strongly in the pharyngeal arches, that were bred to carry the Dfl deletion on one chro head region and limb buds in both chick and mouse mosome 16 and a reciprocal duplication on the other, embryos.38,71 Ufdll was identified as a downstream which restored normal gene dosage. This genetic com gene of a bHLH transcription factor, dHAND, and was plementation examination suggested that the aortic downregulated in dHAND-null mouse embryos that arch anomalies observed in Dfl/+ mice were due to display pharyngeal arch hypoplasia and cardiac outflow haploinsufficiency of a gene(s) mapped within the Dfl tract defects.38 Antisense attenuation of Ufdll in chick region. Mice with a hemizygous deletion of •`150kb of cardiac neural crest cells leads to increased incidence of the proximal region of the TDR, which encompasses 7

septation failure of cardiac outflow tract (unpublished genes, and mice with a hemizygous deletion of a 550kb observation, 2001). These observations suggest that region, which encompasses 16 genes partially over HIRA and UFD1L may contribute to some aspects of lapping Dfl were subsequently generated by Kimber et the 22q11DS phenotype. However, mice heterozygous al. and Puech et al., respectively, but neither of them for either Hira or Ufdll mutation are apparently normal showed any phenotype of 22q11DS.78.79 Later, mice and homozygotes die during early embryogenesis.12.73 with hemizygous deletion from Idd to Hira (Lgdel/+), a Crkol (CRKL in human) encodes an SH2-SH3-SH3 region containing the Dfl/+ region plus 5 more genes,

adapter protein involved in growth-factor response and generated by Merscher et al., exhibited similar types of focal-adhesion signaling, and is expressed in tissues aortic arch anomalies as Dfl/+ in about 50% of cases, derived from neural crest cells during development.74 as well as parathyroid gland aplasia.80 Lindsay et al. Mice homozygous null for Crkol are embryonic lethal, and Merscher et al. independently narrowed the criti and develop a similar phenotype to that of 22q11DS, cal region using chromosomal engineering and rescue including conotruncal and aortic arch defects, thymus transgenesis, and came to the same conclusion that Tbxl and craniofacial abnormalities, suggesting a potential is responsible for the aortic arch phenotype observed in contribution of CRKL to the 22q11DS phenotype.74 Dfl/+ and Lgdel/+ mice. Further establishment and However, mice heterozygous for Crkol mutation are analysis of mice heterozygous for Tbxl mutation alone normal and viable, indicating that Crkol is not haplo by both groups supported this conclusion.72.80 insufficient, at least in the mouse. COMT, which encodes catecol-O-methyltransferase TBX1: TBX1 belongs to the T-box family of tran and PRODH, which encodes proline dehydrogenase, scription factors.81 Members of this family are involved are potentially involved in the behavioral and psychiat in early development of both vertebrates and inverte ric phenotype of 22q11DS.75-77 Comt mutant mice dis brates, including specification of mesoderm and endo play impairment in emotional behavior. Prodh mutant derm, and heart and limb morphogenesis (Reviewed in mice have a deficit in sensorimotor gating that is a 82). Jerome and Pappaioannou independently reported neural filtering process, that allows attention to be fo mice homozygous and heterozygous for a Tbxl muta tion allele.83 According to their report cused on a given stimulus, and is affected in patients , mice homo with some psychiatric disorders. zygous for Tbxl mutation died perinatally and displayed virtually all testable features of 22q11DS , including conotruncal defects, abnormal facial features Approach using genomic engineering in the mouse: , cleft Recent advanced genome manipulation technologies in palate, and hypoplasia of the thymus and parathyroid mice have more clearly provided an insight into the role glands. Mice heterozygous for Tbxl mutation have only of candidate genes for 22q11DS.47 The human region aortic arch patterning defects with a less penetrant on chromosome 22q11 and the syntenic, or orthologous, phenotype, although they do not reflect the entire phe 84 Yamagishi H: The 22q11.2 deletion syndrome

Fig. 4 Expression patterns of Tbxl. 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 Tbxl 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 primary neural crest culture cells, HNK-1 (G) and neuropilin-1 (H) are detected by immunocytochemistryand mRNA in situ hybridization, respectively, but Tbxl is not detectable by mRNA in situ hybridization (I). fg, forgut; da, dorsal aorta; ht, heart.

notypic spectrum of 22q11DS. These results provided endodermal epithelium of pharyngeal arches as well evidence for TBX1 likely being a major genetic deter as in the head mesenchyme. Whole mount and section minant in the etiology of 22q11DS. Importantly, mice in situ hybridization using chicken Tbxl riboprobe heterozygous for Tbxl showed a varying degree of demonstrated a similar expression pattern to that of absent or reduced 4th pharyngeal arch arteries, later mouse Tbxl. Interestingly, Tbxl was not detectable in resulting in aortic arch anomalies, depending on their neural crest-derived mesenchyme in the pharyngeal genetic background. This phenotype nicely matches arches of both mouse and chicken embryos. The ex the abnormalities observed in Dfl/+ and Lgdel/+ pression analysis of Tbx1 in the primary chicken neural mice, consistent with the analyses of the two other crest cells further clearly demonstrated that Tbxl was groups. 72,80,83However, mutational analysis of the not expressed in neural crest cells. coding region of TBX1 in over 200 patients with a clin ical suspicion of 22q11DS but without deletion or other Future directions detectable rearrangements has so far failed to identify TBX1 gene mutations.72,84 A finding that TBX1 haploinsufficiency is likely to be To determine the relevance of Tbxl to the patho the major determinant of aortic arch defects in patients genesis of 22q11DS, we performed detailed expression with 22q11DS is a significant advancement in this field. analyses of mouse and chicken Tbxl in pharyngeal arch The pathogenetic basis of 22q11DS would become development (Fig. 4).85 In mouse embryos at embry more clear by future efforts to identify the genes that onic day (E) 9.5, Tbxl was preferentially expressed in are targets of TBX1, to define molecular pathways the pharyngeal arches, the ventral half of the otic vesi controlling TBX1 expression, and to elucidate the role cle, and in the head. Section in situ hybridization at of TBX1 in cardiovascular and pharyngeal arch de E10.5 showed expression of Tbxl in the mesodermal velopment. Although functional mutations have not core, adjacent to the pharyngeal arch arteries, and the been identified in patients with a clinical suspicion of Keio J Med 2002; 51 (2): 77-88 85

22q11DS but without the deletion, further extensive conserved in mice. mutational analyses are indicated for coding, non cod Finally, the research advances described here should ing and regulatory regions of TBX1. be beneficial to patients with 22q11DS. The continuous The delineation of the expression pattern of Tbxl efforts to delineate the clinical features with accurate sheds light on the molecular and cellular basis of prevalence will lead to effective evaluation and inter 22q11DS. Interestingly, Tbxl is strongly expressed in vention for the patients. Better knowledge for the be endoderm and mesoderm cells, but not in neural crest havioral and psychiatric phenotype and the phenotype cells during the pharyngeal arch development.81,83,85 in adult age need to be accumulated over the next This result suggests that the primary lesion of the main few years. One of the ultimate goals for basic research features in 22q11DS may not be the neural crest cells, is preventive treatment (and eventual gene therapy) although 22q11DS has been believed to result primarily of afflicted infants after discovery of the responsible frrbnormal development of neural crest cells as gene(s) and their function, although it requires much mentioned above. Numerous recent studies have dem more progress to achieve these interventions. Searching onstrated that appropriate epithelial-mesenchymal sig for the upstream or downstream targets of Tbxl may naling is essential for proper development of the pha be the next key step toward the goal. A multifaceted ryngeal arches, and that the primary impairment of the approach to dissect the clinical and genetic aspects of epithelial endoderm can secondarily affect migration or 22q11DS would develop a deeper understanding and differentiation of neural crest cells during the pharyn a better intervention for patients with 22q11DS or the geal arch development.86-88 Importantly, mutations variety of disorders associated with this syndrome. in genes expressed in either neural crest cells or non neural crest cells in pharyngeal arches can result in Acknowledgments:The author wishesto thank the patient and his similar aortic arch defects in mice. Future studies are familyfor the photograph(the permissionfrom the familyhas been thus required to focus on the signals that mediate inter obtained,and the permissionfor the use of the photographin this publicationhas been obtained from the family). The author also actions between neural crest cells and endoderm cells wishes to thank Dr. Deepak Srivastavafor critical reading of the and/or mesoderm cells in pharyngeal arches to uncover manuscript,Dr. ChihiroYamagishi for preparationof the figures,and the developmental mechanism of 22q11DS. Dr. YoshifumiKojima and membersof Divisionof PediatricCardi Although Tbxl haploinsufficiency accounts for the ology in Keio UniversitySchool of Medicine,and Prof. Nobutake Matsuofor their support.Expression analyses of Tbx1was performed aortic arch defects in Dfl/+ mice, haploinsufficiency of in Dr. Srivastava'slaboratory at Universityof Texas Southwestern TBX1 may not be the only cause of the entire clinical MedicalCenter. presentations of 22q11DS. Dfl/+ mice have broader phenotypic features that have not been described in mice heterozygous for Tbxl mutation, including con References otruncal defects (overriding aorta plus ventricular 1. Scambler PJ: The 22q11 deletion syndromes. 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