Congenital heart defects and 22q11 deletions: which genes count?

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MOLECULAR MEDICINE TODAY, AUGUST 1998

Congenital heart defects and 22q11 deletions: which genes count? Elizabeth A. Lindsay and Antonio Baldini

Hemizygous deletions on the long arm of chromosome 22 (del22q11) are a relatively common cause of congenital heart disease. For some specific heart defects such as interrupted aortic arch type B and tetralogy of Fallot with absent pulmonary valve, del22q11 is probably the most frequent genetic cause. Although extensive gene searches have been successful in discovering many novel genes in the deleted segment, standard positional cloning has so far failed to demonstrate a role for any of these genes in the disease. We show how the use of experimental animal models is beginning to provide an insight into the developmental role of some of these genes, while novel genome manipulation technologies promise to dissect the genetic aspects of this complex syndrome. DELETIONS of the chromosome region 22q11.2 have an estimated prevalence of 1:4000 live births1, making them one of the most frequent causes of genetic disease. Most are sporadic in origin, but a recent large study of affected individuals found that 28% had inherited the deletion from an affected parent2. The deletion is associated with two distinct but related clinical presentations, namely DiGeorge syndrome (DGS) and velocardiofacial syndrome (VCFS, also known as conotruncal anomaly face syndrome*). However, del22q11 is often associated with an incomplete clinical picture. The cardinal features of DGS are: absence or hypoplasia of the thymus and parathyroid glands, which causes immune defects and seizures, respectively; congenital heart defects (CHDs); facial anomalies; and mild to moderate mental retardation. VCFS has some of the above features, but can be distinguished clinically by the characteristic facial phenotype and generally less severe forms of CHD. The diagnosis of VCFS is often made in older children or young adults, as important diagnostic features may not be present in infancy, such as the characteristic facial

Elizabeth A. Lindsay PhD Research Associate Antonio Baldini* MD Assistant Professor Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, T936, Houston, TX 77030, USA. Tel: +1 713 798 6519 Fax: +1 713 798 5386 *e-mail: [email protected]

features, learning difficulties and psychiatric disorders. Features of both syndromes can also occur in the same patient. In this review, we will refer to the 22q11 phenotype to indicate the complex clinical spectrum associated with 22q11 deletions. Table 1 summarizes the main clinical features associated with the del22q11 phenotype and is derived from a European collaborative study of 558 deleted patients from centers in seven European countries2. A striking aspect of the del22q11 phenotype is its variability, the basis of which is not understood. It cannot be related to deletion size for the following reasons: (1) most patients apparently have the same deletion, estimated to be about 2 megabases; (2) there is intrafamilial phenotype variability, although affected family members have presumably inherited identical deletions; (3) genotypeÐphenotype correlations cannot be made (i.e. smaller deletions do not result in milder or more restricted phenotypes). Suggested explanations for the variable phenotype include allelic variability, variable penetrance and variable expressivity caused by environmental factors or stochastic events during fetal development. Similar phenotype variability occurs in other genetic syndromes caused by microdeletions, such as Williams syndrome and PraderÐWilli syndrome**.

22q11 deletions and congenital heart defects The types of CHDs found in del22q11 patients are conotruncal defects and aortic arch anomalies (Fig. 1). Conotruncal anomalies include truncus arteriosus (failure of septation of the primitive single *McKusick Index 188400 and 192430, respectively; McKusick, V. On line Mendelian Inheritance in Man, http://www3.ncbi.nlm.nih.gov/Omim/ **McKusick Index 194050 and 176270, respectively; McKusick, V. On line Mendelian Inheritance in Man, http://www3.ncbi.nlm.nih.gov/Omim/

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Lcc

Table 1. The del22q11 phenotype Phenotype in del22q11 patients

Subc

Frequency of occurrencea (%)

Classical features Congenital heart defects

AA 75 (n = 545)

Mild to moderate immune deficiency

66 (n = 218)

Severe immune deficiency

1.4 (n = 218)

Hypocalcemia

60 (n = 340)

Facial anomalies

‘Majority’

Developmental delay

68 (n = 338)

PA

LA

Additional common features Height/weight <50th percentile

83 (n = 158)

Cleft palate

9 (n = 496)

Velopharyngeal insufficiency

32 (n = 496)

Psychiatric disorders as adults

18 (n = 61)

Renal defects

36 (n = 136)

Skeletal defects

17 (n = 548)

Hearing defects

33 (n = 159)

Ao

RA LV RV

a

Data derived from Ref. 2.

vessel from the heart into the aorta and pulmonary artery), tetralogy of Fallot and double outlet right ventricle (caused by malalignment of the aorta with respect to the ventricular septum), ventricular septal defects (caused by incomplete development of the conotruncus), pulmonary atresia and d-transposition of the great vessels. Aortic arch anomalies include interrupted aortic arch (IAA) type B, coarctation of the aorta and right aortic arch. Several studies have assessed the frequency of CHDs in del22q11 patients (reviewed in Ref. 3 and summarized in Table 2). The five heart defects listed in Table 2 are those most commonly found in del22q11 patients and account for about 85% of CHDs in these patients. It is clear from these data that the frequency of certain CHDs is much higher in del22q11 patients than in CHD patients generally; for example, IAA type B occurs in an average of 17% of del22q11 patients, compared with <1% of cases of critical CHDs in infants in the general population. Other studies have assessed the frequency of 22q11 deletions in patients ascertained because of their heart defects. Two prospective studies of patients with conotruncal and aortic arch anomalies found 22q11 deletions to be present in 11Ð17% of patients4,5. For specific heart defects, the frequency of deletions was much higher (Table 3). For example, 50% of patients with IAA type B carried the deletion, irrespective of the presence of other features of del22q11 syndrome4Ð6. Thus, a specific genetic defect accounts for the etiology of IAA type B in 50% of cases, making it one of the most etiologically homogeneous CHDs. Other CHDs that are associated with a high incidence of deletion are tetralogy of Fallot in association with pulmonary atresia or with absent pulmonary valve (Table 3). The developmental field defect underlying the del22q11 phenotype is thought to result from defects of neural crest cell migration or function during early embryonic life. This attribution derives from two sources: (1) the observation that the anatomical structures af-

Figure 1. Diagram of an adult heart. The regions most commonly affected in del22q11 syndrome (the conotruncus and part of the aortic arch) are highlighted in red. AA, aortic arch; PA, pulmonary artery; LA, left atrium; RA, right atrium; RV, right ventricle; LV, left ventricle; Ao, aorta; Lcc, left common carotid artery; Subc, left subclavian artery.

fected in del22q11 (heart outflow tract, thymus, parathyroids, mandible and maxilla) derive from the cephalic neural crest (reviewed in Ref. 10), and (2) experiments on chick embryos, where ablation of cardiac neural crest cells before their migration from the neural fold results in a del22q11-like phenotype (reviewed in Ref. 11). The cardiac neural crest, a subpopulation of the cephalic neural crest, originates in the midotic placode to somite 3 region of the developing embryo. Cardiac neural crest cells migrate from the neural tube through the circumpharyngeal region to populate the caudal pharyngeal arches (IIIÐVI), the aortic arch and the cardiac outflow tract, where they play an essential role in the development of the aortic arch and in aorticopulmonary septation. Ablation of the cardiac neural crest in chick embryos, before its migration from the neural fold, leads to heart abnormalities reminiscent of those found in del22q11 patients, namely IAA, truncus arteriosus, tetralogy of Fallot, double outlet right ventricle and ventricular septal defects. Most of our knowledge of the neural crest derives from avian studies, and the existence of an equivalent cell lineage in mammals had been presumed but not proven until recently. Its existence and its role in normal cardiac development in mammals has been elegantly 351

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of their predicted function or suggestive developmental expression pattern. Historically, the first gene shown to be deleted in del22q11 patients was HIRA (Refs Heart defect Incidence in 652 del22q11 Prevalence in unselected 18,19). This gene has been proposed to be patients with CHDsa (%) CHD patientsb (%) involved in chromatin assembly based on high sequence similarity with Hir1p and Interrupted aortic arch type B 17.2 <1 Hir2p of Saccharomyces cerevisiae and with Truncus arteriosus 10.4 2–4 the p60 subunit of human chromatin assemTetralogy of Fallot 25.5 8 bly factor 1 (CAF1). Hir1p and Hir2p are repressors of histone gene transcription, while Ventricular septal defect 17.0 20 CAF1 is involved in histone assembly onto Pulmonary atresia 14.4 3 newly synthesized DNA. It is hypothesized aData derived from Ref. 2. that a reduced HIRA dosage in deleted pabAdapted from Ref. 56. tients could affect normal gene expression CHD, congenital heart defect. via altered chromatin assembly. Expression analysis of HIRA during mouse and chick embryogenesis shows broad expression from Table 3. Prevalence of 22q11 deletions in gastrulation onwards, and is hence compatible with a possible role of this gene in patients with selected congenital heart defects normal development20. A member of the paired-like class of Heart defecta Prevalence of 22q11 deletion Refs homeobox genes, goosecoid-like (GSCL)21, IAA type B 15/30 (50%) 4–6 derives its name from the highly similar gene goosecoid (GSC). GSC, a transcription reguTOF with pulmonary atresia 14/33 (42%) 4,7 lator, is essential for normal craniofacial and TOF with absent pulmonary valve 7/11 (64%) 4,6,8,9 rib-cage development in the mouse22,23. Based on the similarity to GSC, it was specuaData derived from references listed. lated that GSCL might contribute to the craIAA, interrupted aortic arch; TOF, tetralogy of Fallot. niofacial phenotype of del22q11 patients. However, expression of GSCL during mouse demonstrated by Conway et al.12, using the splotch mouse. Splotch embryogenesis suggests that GSC and GSCL have distinct developmice carry a mutation in the Pax3 gene, and splotch homozygotes mental roles, as expression was detected in the developing pons but have conotruncal heart defects and thymus, parathyroid and thyroid not in developing craniofacial structures24. defects. Conway et al. have demonstrated that Pax3 can be used as a The gene T-Box1 (TBX1) is one of seven known genes that all marker for cardiac neural crest cells in mouse embryos and, tracing share a highly conserved DNA-binding domain (T-box), and are cardiac neural crest cells from the neural tube to the heart outflow named after the gene mutated in the mouse mutant Brachyury (T) tract in normal and splotch mice, they demonstrated that, in splotch (reviewed in Ref. 25). The mouse homolog of TBX1 is expressed in embryos, cardiac neural crest cells migrated normally from the the pharyngeal arches and pouches, in the otic vesicle and in the deneural tube but failed to reach the pharyngeal arches and heart out- veloping spinal column26. Based on this pattern of expression, it was flow tract. Their work is the first direct demonstration in mammals of speculated that TBX1 might contribute to some of the phenotypic a conotruncal heart defect that results from a failure of neural crest features associated with del22q11, including the frequent hearing migration. In the future, when mouse models of del22q11 syndrome defects and skeletal anomalies2. Members of this interesting gene become available, markers of cardiac neural crest cells might eluci- family are differentially expressed during mouse embryogenesis26. Mutations of TBX5 have been shown recently to be responsible for date the role of this cell lineage in del22q11 syndrome. HoltÐOram syndrome, a developmental disorder affecting the heart The deleted genes and upper limbs14,15. Extensive gene searches and genomic sequencing have identified 18 ZNF74 (Ref. 27) is a member of the Cys2/His2 class of zinc-finger genes in the deleted region (Fig. 2), which have been reviewed genes. The protein products of these genes contain a highly conrecently13. Excluded from this list are transcripts without obvious served KrŸppel-associated box (KRAB) domain, which is a trancoding potential or genes that are not completely characterized. In scription repressor motif. ZNF74, which is expressed only in fetal this review we will mainly discuss transcription factors. This is tissues, codes for an RNA-binding protein associated with the nuthe most well-represented category of genes in the region, with six of clear matrix, and has been shown to interact with the hyperphosthe deleted genes coding for putative transcription factors. phorylated largest subunit of RNA polymerase II. Overall, these findTranscription factors have been involved in a number of develop- ings led to speculation that ZNF74 might be involved in the mental defects, including CHDs in human disease, for example developmental regulation of pre-mRNA processing and, thereby, in HoltÐOram syndrome (TBX5)14,15, Waardenburg syndrome (Pax3)16 the regulation of gene expression. and X-linked situs inversus (ZIC3)17. In addition, our discussion will Finally, LZTR1 encodes a protein with a basic leucine zipper doinclude some of the other genes considered to be of interest because main that, along with other sequence characteristics, makes it a good

Table 2. The most common heart defects in del22q11 patients

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transcription regulator candidate. Northern blot analysis reveals that it is expressed in Gene symbols/ Description / predicted or known function aliases several human fetal tissues28, and it would be Centromere interesting to study the spatiotemporal exHomologous to Drosophila gonadal, unknown function DGCR6 pression of the homologous mouse gene durIDD/DGCR2 Putative transmembrane protein ing mouse embryogenesis. ADU Among the genes not known to be inTSK/DGS-G Testes-specific threonine-serine kinase volved in transcription regulation, three are ES2/DGS-I Highly conserved nuclear protein, unknown function of potential interest. IDD/DGCR2 (Refs 29,30) G codes for a putative transmembrane protein GSCL Goosecoid-like homeobox gene that has similarity to the ligand-binding doCTP Putative citrate transporter protein main of the low-density lipoprotein receptor 2+ and to the C-type lectins, which are Ca CLTCL Putative clathrin heavy chain-like polypeptide GM00980 dependent carbohydrate-binding proteins. HIRA WD-40-containing, putative transcription regulator These similarities suggest that IDD has a GM05878 TMVCF Putative transmembrane protein role in cell adhesion. More specifically, cellsurface glycoproteins such as C-lectins have UFD1L Putative ubiquitination fusion degradation protein been shown to affect neural crest migration, CDCrel-1 Putative member of septin gene family both in vitro and in vivo. Expression analysis GM05401 of the mouse homolog of IDD shows that it GP Ibβ Platelet glycoprotein, β subunit is ubiquitously expressed during early mouse TBX1 T-box-containing putative transcription factor embryogenesis and is not restricted to cells T10 Unknown function of neural crest origin31. ES2/DGS-I (Refs 32,33) is a highly conCOMT Catechol-O-methyltransferase served gene with homologs in species as ARVCF Armadillo repeat-containing gene, unknown function far back as Caenorhabditis elegans and Drosophila, but the protein encoded by ES2 LZTR-1 Putative transcription factor does not show any similarities with other ZNF74 Zinc finger-containing gene proteins of known function. The mouse homolog is expressed widely in early mouse development, with higher levels of expression in the pons, in a region apparently idenFigure 2. Eighteen genes have been identified in the deleted region and their physical order on the chrotical to that expressing GSCL (Ref. 24). mosome has been established. Colored symbols identify genes that map within key patient breakpoint inFigure 3 shows the expression of ES2 and tervals. Black horizontal lines indicate patient breakpoints38. Black horizontal zig-zags represent the boundaries of the common deletion. The drawing is not to scale. GSCL in the developing mouse pons. CLTCL (Refs 34Ð36) has high similarity to the clathrin heavy chain gene that maps to chromosome 17. Clathrin is the major protein component of clathrin- tification of subsegments that are more consistently deleted; these are coated pits and vesicles involved in receptor-mediated endocytosis, termed Ôcritical regionsÕ38Ð40. However, we must be cautious when whereby vesicles are transported from the plasma membrane and the using this term, as some of the genetic lesions in del22q11 do not trans-Golgi network to the lysosomes. The role of this protein in overlap, even though the associated phenotypes do. Three different such basic cell functions as intracellular transport and signal trans- models could explain this scenario: (1) A single large gene is disrupted by all these patient breakduction made the closely related CLTCL an exciting candidate gene for a role in the del22q11 phenotype. However, surprisingly, CLTCL points. Despite extensive gene searches and genomic sequencing, no appears to be a recently evolved heavy chain gene and no mouse hom- such hypothetical gene has been found so far. (2) Multiple genes affecting the same developmental pathway olog has been found to date, in contrast to the clathrin heavy chain gene located on chromosome 17, which is highly conserved. The pu- might be present in the region. At this time, as we do not know which tative CLTCL peptide is truncated at the COOH terminus in compari- developmental pathway is affected by del22q11, this hypothesis is son with other clathrin heavy chain peptides, but it is not known impossible to test. (3) The different chromosomal breakpoints are affecting the norwhether this has functional significance. The report of a balanced translocation breakpoint interrupting CLTCL (Ref. 37) in a patient mal regulation/expression of the same genes, although not directly with only a few minor characteristics in common with the del22q11 disrupting them. This is an intriguing hypothesis that includes the syndrome suggests that this gene is unlikely to be a major player in possibility of the presence of a locus control region, regulatory elements controlling multiple genes in the region and/or alterations of this syndrome. chromatin structure affecting gene regulation over a long distance. Which gene counts? This hypothesis does not exclude the possibility that critical genes Given the large number of genes identified in the deleted region, it for this syndrome are located outside the deleted region. would be desirable to be able to select a subset for further studies. Is del22q11 a contiguous gene syndrome? The concept of a conExtensive analysis of patient deletion breakpoints has led to the iden- tiguous gene syndrome was first proposed by Schmickel41 and refers 353

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Figure 3. The genes GSCL (a) and ES2 (b) are coexpressed in the pons during early mouse development. This is shown here by RNA in situ hybridization on sagittal sections of 11.5-day mouse embryos.

Glossary Chromosome engineering – A strategy by which chromosomes can be artificially modified, according to a specific design, to generate deletions, duplications or translocations. When this is performed using mouse embryonic stem cells, the phenotypic effect of the chromosomal modifications can be studied in vivo. Comparative mapping – Physical mapping to establish the chromosomal location and relative order of groups of genes or DNA markers in different species, such as human and mouse. Conotruncus – Anterior portion of the primitive heart tube, which, at the end of cardiac looping, forms the ventricular outflow tract. Contiguous gene syndrome – A clinically recognizable syndrome caused by deletion or duplication of a DNA segment containing multiple genes. The extent of the deletion/duplication correlates with the phenotype, and individual features of the phenotype might be inherited in isolation. Developmental field – A group of embryonic cells that develop together in a coordinated manner and react to environmental or genetic factors/insults as a group. Haploinsufficiency syndrome – Syndrome resulting from the loss of function of one allele of a gene, despite the presence of the protein product of the remaining normal allele. Kallmann syndrome – A genetic syndrome characterized by hypogonadotrophic hypogonadism and anosmia (no sense of smell). It is mostly inherited as an X-linked disorder, but cases of autosomal-recessive and autosomal-dominant inheritance have been reported. Knockout mouse – Mouse carrying a targeted mutation in a specific gene, which renders that gene non-functional. The targeted mutation is made in vitro in mouse embryonic stem (ES) cells by homologous recombination. The ES cells are introduced into mouse embryos where they populate tissues, including the germline. This allows the mutation to be transmitted to the progeny. Midotic placode – One of a group of ectodermal thickenings in the head region of the embryo. The otic placode gives rise to the vestibulocochlear ganglion and parts of the inner ear. Positional cloning – A strategy by which a disease gene is identified by virtue of its map position on the chromosome, rather than for its function. Syntenic genes – Genes that are located on the same chromosome. Tetralogy of Fallot – A congenital heart defect characterized by a large ventricular septal defect, right ventricular outflow obstruction, dextroposition of the aorta and right ventricular hypertrophy.

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to conditions resulting from the deletion (or duplication) of a DNA segment containing Normal chromosome multiple genes. Different genes contribute to a b c d e different aspects of the phenotype, and different deletions generate subsets of the pheInsertion of two loxP sites notypic spectrum caused by larger deletions. Classical examples are deletions involving the loxP loxP short arm of the X chromosome, which can cause chondrodysplasia punctata, steroid sulfaa b c d e tase deficiency and Kallmann syndrome phenotypes in isolation or in combination accordCre expression ing to the extent of the deletion42. Although it is possible that multiple genes contribute to the a b del22q11 phenotype, it is not apparent that difd e ferent deletions cause different subsets of the clinical spectrum; in fact, phenotypes tend to c be similar with different deletions. The phenotype of a microdeletion synRecombination products drome can be due to haploinsufficiency of a single gene, or of multiple genes. If the forDeleted chromosome mer is suspected, a common strategy is to a e identify ÔtypicalÕ patients lacking the deletion and to search for single gene mud b tations within the locus usually deleted. This Circular fragment strategy has been successful in a number of (lost because acentric) 43,44 cases, including Angelman syndrome and c Alagille syndrome45,46, but it has failed so far in del22q11 patients29,30,32,47,48. This could be Figure 4. Schematic of the Cre–lox strategy used to generate large genomic deletions. Two loxP sites are because the ÔrightÕ gene has not yet been inserted at the desired deletion endpoints in the mouse chromosome by homologous recombination (like those used to generate gene knockouts). Subsequently, transient expression of the Cre protein causes tested, or because the Ôtypical phenotypeÕ recombination between the loxP sites with loss of the DNA between the two loci. cannot be produced by a single gene mutation. Furthermore, the etiological heterogeneity of the del22q11-like phenotype rePerhaps the most important use of an animal system will be the duces the likelihood of success of this approach. For example, a similar phenotype can be caused by deletions of the short arm of generation of a model reproducing the common human genetic lechromosome 10 (reviewed in Ref. 49), alcohol or retinoic acid sion. It is undeniable that animal models of human diseases can substantially help our understanding of pathogenetic mechanisms50. This consumption during pregnancy, and maternal diabetes. The identification of a single gene mutation in patients without is especially true for developmental defects originating in early emdeletions would represent tremendous progress in the study of the bryonic life. For obvious reasons, these particular defects cannot be del22q11 syndrome. However, this finding will not address the com- studied in great detail in humans. The generation of mammalian plexity of the genetic lesion of the great majority of patients who, by models of single gene defects has been very successful, thanks to definition, are deleted for a large number of genes. technologies allowing the targeted manipulation of mouse genes in embryonic stem (ES) cells. Manipulated ES cells can be injected into Assistance from mice early mouse embryos where they differentiate and populate the variOwing to the genetic complexity of the del22q11 syndrome and the ous tissues and organs, including the germline. As a result, at the next difficulty of conducting molecular genetics studies during human embryonic development, several investigators are trying to develop a mouse model. These efforts have been initiated by the isolation of murine homologs of the genes deleted in del22q11 syndrome and the The outstanding questions study of their expression during mouse embryonic development. Is del22q11 a single or multiple gene defect? Developmental expression studies can suggest a possible role of Are any of the genes identified so far in the deleted region genes in the development of organs and systems. Several genes have relevant for the phenotype? been studied in this way and we have briefly reviewed the results in What is the developmental pathway(s) affected by the section on deleted genes. Remarkably, no gene specifically exdel22q11? pressed in developing heart has been found so far. However, a numWhat is the basis of the phenotype variability? ber of genes are broadly expressed during early embryogenesis and Is there a specific gene in the deleted region involved in hence are compatible with a role in heart development. Alternatively, heart development? the Ôcritical gene(s)Õ might have a pleiotropic effect on early development rather than on specific organs and systems.

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generation, the manipulated gene can be transmitted to the progeny, and mice carrying the mutation in all the somatic tissues can be generated. By contrast, models of chromosomal syndromes (i.e. those involving large segments of DNA as opposed to single genes) are very difficult to obtain because (1) until recently, there was no suitable technology for the generation of precisely targeted rearrangements, and (2) the group of genes located on a large human genomic segment may not be syntenic in mice. Recently, the first problem has been resolved and methods to generate targeted chromosomal rearrangements in ES cells, including large chromosomal deletions and translocations, are available51,52. The technology takes advantage of a bacteriophage recombinase, Cre, that catalyzes recombination between special short sequences, named loxP. A scheme of this procedure is illustrated in Fig. 4. In order to apply this technology to the del22q11 region, investigators have asked whether there is a genomic locus in mouse comparable in gene content to the human del22q11 locus. Galili et al.53 have reported the DNA sequence of a 38 kb segment of mouse chromosome 16 that has remarkable similarity to a segment of the del22q11 locus, and, more recently, extensive comparative mapping data show that the murine homologs of virtually all the genes known to be deleted in del22q11 are located within the chromosome 16 mouse locus54,55. However, the order of the genes is different from that in humans and one gene (CLTCL) could not be found in mouse. Overall, comparative mapping data are highly encouraging and a targeted deletion approach in mouse should be considered feasible.

Concluding remarks del22q11 contributes to a significant number of critical congenital heart disease cases, as well as to other birth defects. Deletion breakpoint mapping has demonstrated the genetic complexity of this syndrome and, combined with the large number of genes involved, this makes del22q11 syndrome a major challenge for geneticists. The use of animal systems and new genome manipulation technologies appear to be the most promising approaches for detailed genetic dissection of this condition and for the identification of developmental pathways and genes affecting normal heart development. References 1 Wilson, D.I. et al. (1994) Minimum prevalence of chromosome 22q11 deletions, Am. J. Hum. Genet. Suppl. 55, A975 2 Ryan, A.K. et al. (1997) Spectrum of clinical features associated with interstitial chromosome 22q11 deletions: a European collaborative study, Med. Genet. 34, 798Ð804 3 Lewin, M.B., Lindsay, E.A. and Baldini A. (1996) 22q11 deletions and cardiac disease, Prog. Pediatr. Cardiol. 6, 19Ð28 4 Webber, S.A. et al. (1996) Importance of microdeletions of chromosomal region 22q11 as a cause of selected malformations of the ventricular outflow tracts and aortic arch: a three-year prospective study, J. Pediatr. 129, 26Ð32 5 Lewin, M.B. et al. (1997) A genetic etiology for interruption of the aortic arch type B, Am. J. Cardiol. 80, 493Ð497 6 Mehraein, Y. et al. (1997) Microdeletion 22q11 in complex cardiovascular malformations, Hum. Genet. 99, 433Ð442 7 Digilio, M.C. et al. (1996) Comparison of occurrence of genetic syndromes in ventricular septal defect with pulmonic stenosis (classic tetralogy of Fallot) versus ventricular septal defect with pulmonic atresia, Am. J. Cardiol. 77, 1375Ð1376 8 Johnson, M.C. et al. (1995) Deletion within chromosome 22 is common in

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patients with absent pulmonary valve syndrome, Am. J. Cardiol. 76, 66Ð69 9 Amati, F. et al. (1995) 22q11 deletions in isolated and syndromic patients with tetralogy of Fallot, Hum. Genet. 95, 479Ð482 10 Lammer, E.J. and Opitz, J.M. (1986) The DiGeorge anomaly as a developmental field defect, Am. J. Med. Genet. Suppl. 2, 113Ð127 11 Kirby, M.L. and Waldo, K.L. (1995) Neural crest and cardiovascular patterning, Circ. Res. 77, 211Ð215 12 Conway, S.J., Henderson, D.J. and Copp, A.J. (1997) Pax3 is required for cardiac neural crest migration in the mouse: evidence from the splotch (Sp2H) mutant, Development 124, 505Ð514 13 Budarf, M.L. and Emanuel, B.S. (1997) Progress in the autosomal segmental aneusomy syndromes (SASs): single or multi-locus disorders? Hum. Mol. Genet. 6, 1657Ð1665 14 Basson, C.T. et al. (1997) Mutations in human TBX5 cause limb and cardiac malformation in HoltÐOram syndrome, Nat. Genet. 15, 30Ð35 15 Li, Q.Y. et al. (1997) HoltÐOram syndrome is caused by mutations in TBX5, a member of the Brachyury (T) gene family, Nat. Genet. 15, 21Ð29 16 Baldwin, C.T., Hoth, C.F., Macina, R.A. and Milunsky, A. (1995) Mutations in PAX3 that cause Waardenburg syndrome type I: ten new mutations and review of the literature, Am. J. Med. Genet. 58, 115Ð122 17 Gebbia, M. et al. (1997) X-linked situs abnormalities result from mutations in ZIC3, Nat. Genet. 17, 305Ð308 18 Halford, S. et al. (1993) Isolation of a putative transcriptional regulator from the region of 22q11 deleted in DiGeorge syndrome, Shprintzen syndrome and familial congenital heart disease, Hum. Mol. Genet. 2, 2099Ð2107 19 Lamour, V. et al. (1995) A human homolog of the S. cerevisiae HIR1 and HIR2 transcriptional repressors cloned from the DiGeorge syndrome critical region, Hum. Mol. Genet. 4, 791Ð799 20 Wilming, L.G. et al. (1997) The murine homologue of HIRA, a DiGeorge syndrome candidate gene, is expressed in embryonic structures affected in human CATCH22 patients, Hum. Mol. Genet. 6, 247Ð258 21 Gottlieb, S. et al. (1997) The DiGeorge syndrome minimal critical region contains a goosecoid-like (GSCL) homeobox gene that is expressed early in human development, Am. J. Hum. Genet. 60, 1194Ð1201 22 Rivera-Perez, J.A. et al. (1995) Goosecoid is not an essential component of the mouse gastrula organizer but is required for craniofacial and rib development, Development 121, 3005Ð3012 23 Yamada, G. et al. (1995) Targeted mutation of the murine goosecoid gene results in craniofacial defects and neonatal death, Development 121, 2917Ð2922 24 Lindsay, E.A., Harvey, E.L., Scambler, P.J. and Baldini, A. (1998) ES2, a gene deleted in DiGeorge syndrome, encodes a nuclear protein and is expressed during early mouse development, where it shares an expression domain with a Goosecoid-like gene, Hum. Mol. Genet. 7, 629Ð635 25 Papaioannou, V.E. (1997) T-box family reunion, Trends Genet. 13, 212Ð213 26 Chapman, D.L. et al. (1996) Expression of the T-box family genes, Tbx1ÐTbx5, during early mouse development, Dev. Dyn. 206, 379Ð390 27 Aubry, M. et al. (1993) Isolation of a zinc finger gene consistently deleted in DiGeorge syndrome, Hum. Mol. Genet. 2, 1583Ð1587 28 Kurahashi, H. et al. (1995) Isolation and characterization of a novel gene deleted in DiGeorge syndrome, Hum. Mol. Genet. 4, 541Ð549 29 Demczuk, S. et al. (1995) Cloning of a balanced translocation breakpoint in the DiGeorge syndrome critical region and isolation of a novel potential adhesion receptor gene in its vicinity, Hum. Mol. Genet. 4, 551Ð558 30 Wadey, R. et al. (1995) Isolation of a gene encoding an integral membrane protein from the vicinity of a balanced translocation breakpoint associated with DiGeorge syndrome, Hum. Mol. Genet. 4, 1027Ð1033 31 Taylor, C. et al. (1997) Cloning and mapping of murine Dgcr2 and its homology to the Sez-12 seizure-related protein, Mamm. Genome 8, 371Ð375

Reviews

MOLECULAR MEDICINE TODAY, AUGUST 1998

32 Rizzu, P. et al. (1996) Cloning and comparative mapping of a gene from the commonly deleted region of DiGeorge and velocardiofacial syndromes conserved in C. elegans, Mamm. Genome 7, 639Ð643 33 Gong, W. et al. (1996) A transcription map of the DiGeorge and velo-cardiofacial syndrome minimal critical region on 22q11, Mol. Hum. Genet. 5, 789Ð800 34 Lindsay, E.A. et al. (1996) A transcription map in the CATCH22 critical region: identification, mapping, and ordering of four novel transcripts expressed in heart, Genomics 32, 104Ð112 35 Kedra, D. et al. (1996) Characterization of a second human clathrin heavy chain polypeptide gene (CLH-22) from chromosome 22q11, Hum. Mol. Genet. 5, 625Ð631 36 Sirotkin, H. et al. (1996) Isolation of a new clathrin heavy chain gene with muscle-specific expression from the region commonly deleted in velo-cardiofacial syndrome, Hum. Mol. Genet. 5, 617Ð624 37 Holmes, S.E. et al. (1997) Disruption of the clathrin heavy chain-like gene (CLTCL) associated with features of DGS/VCFS: a balanced (21;22)(p12;q11) translocation, Hum. Mol. Genet. 6, 357Ð367 38 Lindsay, E.A. et al. (1993) Molecular cytogenetic characterization of the DiGeorge syndrome region using fluorescence in situ hybridization, Genomics 17, 403Ð407 39 Levy, A. et al. (1995) Interstitial 22q11 microdeletion excluding the ADU breakpoint in a patient with DiGeorge syndrome, Hum. Mol. Genet. 4, 2417Ð2419 40 Kurahashi, H. et al. (1997) Another critical region for deletion of 22q11: a study of 100 patients, Am. J. Med. Genet. 72, 180Ð185 41 Schmickel, R.D. (1986) Contiguous gene syndromes: a component of recognizable syndromes, J. Pediatr. 109, 231Ð241 42 Ballabio, A. et al. (1989) Contiguous gene syndromes due to deletions in the distal short arm of the human X chromosome, Proc. Natl. Acad. Sci. U. S. A. 86, 10001Ð10005 43 Kishino, T., Lalande, M. and Wagstaff, J. (1997) UBE3A/E6-AP mutations cause Angelman syndrome, Nat. Genet. 15, 70Ð73

44 Matsuura, T. et al. (1997) De novo truncating mutations in E6-AP ubiquitinÐprotein ligase gene (UBE3A) in Angelman syndrome, Nat. Genet. 15, 74Ð77 45 Li, L. et al. (1997) Alagille syndrome is caused by mutations in human Jagged1, which encodes a ligand for Notch1, Nat. Genet. 16, 243Ð251 46 Oda, T. et al. (1997) Mutations in the human Jagged1 gene are responsible for Alagille syndrome, Nat. Genet. 16, 235Ð242 47 Gong, W. et al. (1997) Structural and mutational analysis of a conserved gene (DGSI) from the minimal DiGeorge syndrome critical region, Hum. Mol. Genet. 6, 267Ð276 48 Gottlieb, S. et al. (1997) The DiGeorge syndrome minimal critical region contains a goosecoid-like (GSCL) homeobox gene that is expressed early in human development, Am. J. Hum. Genet. 60, 1194Ð1201 49 Shapira, M. et al. (1994) Deletion of the short arm of chromosome 10 (10p13): report of a patient and review, Am. J. Med. Genet. 52, 34Ð38 50 Bedell, M.A., Jenkins, N.A. and Copeland, N.G. (1997) Mouse models of human disease. Part II: recent progress and future directions, Genes Dev. 11, 11Ð43 51 Ramirez-Solis, R., Liu, P. and Bradley, A. (1995) Chromosome engineering in mice, Nature 378, 720Ð724 52 Smith, A.J. et al. (1995) A site-directed chromosomal translocation induced in embryonic stem cells by CreÐloxP recombination, Nat. Genet. 9, 376Ð385 53 Galili, N. et al. (1997) A region of mouse chromosome 16 is syntenic to the DiGeorge, velocardiofacial syndrome minimal critical region, Genome Res. 7, 17Ð26 54 Botta, A., Lindsay, E.A., Jurecic, V. and Baldini, A. (1997) Comparative mapping of the DiGeorge syndrome region in mouse shows inconsistent gene order and differential degree of gene conservation, Mamm. Genome 8, 890Ð895 55 Puech, A. et al. (1997) Comparative mapping of the human 22q11 chromosomal region and the orthologous region in mice reveals complex changes in gene organization, Proc. Natl. Acad. Sci. U. S. A. 94, 14608Ð14613 56 Ferencz, C. and Neill, C.A. (1992) Cardiovascular malformations: prevalence at live birth, in Neonatal Heart Disease (Freedom, R.M., Benson, L.N. and Smallhorn, J.F., eds), pp. 19Ð27, Springer-Verlag

Focus on genetic disease MMT publishes a wide range of articles on the molecular basis of genetic diseases and prospects for their diagnosis and therapy. Here’s a collection of recent reviews on genetic disease… Kirchhausen, T. (1998) WiskottÐAldrich syndrome: a gene, a multifunctional protein and the beginnings of an explanation, Mol. Med. Today 4, 300–304 Young, J. and Povey, S. (1998) The genetic basis of tuberous sclerosis, Mol. Med. Today 4, 313–319 Chavany, C. and Jendoubi, M. (1998) Biology and potential strategies for the treatment of GM2 gangliosidoses, Mol. Med. Today 4, 158–165 Morison, I.M. and Reeve, A.E. (1998) Insulin-like growth factor 2 and overgrowth: molecular biology and clinical implications, Mol. Med. Today 4, 110–115 Parkes, M., Satsangi, J. and Jewell, D. (1997) Mapping susceptibility loci in inflammatory bowel disease: why and how? Mol. Med. Today 3, 546–553 Bates, G.P. and Davies, S.W. (1997) Transgenic mouse models of neurodegenerative disease caused by CAG/polyglutamine expansions, Mol. Med. Today 3, 508–515

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Uitto, J., Pulkkinen, L. and McLean, W.H.I. (1997) Epidermolysis bullosa: a spectrum of clinical phenotypes explained by molecular heterogeneity, Mol. Med. Today 3, 457–465

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Gayther, S.A. and Ponder, B.A.J. (1997) Mutations of the BRCA1 and BRCA2 genes and the possibilities for predictive testing, Mol. Med. Today 3, 168–174

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