European Journal of 53 (2010) 6–13

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European Journal of Medical Genetics

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Genetic factors in , tracheo-esophageal fistula and the VACTERL association: Roles for FOXF1 and the 16q24.1 FOX gene cluster, and review of the literature

Charles Shaw-Smith a,b,* a Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK b Institute of Child Health, 30, Guilford Street, London WC1N 1EH, UK article info abstract

Article history: Esophageal atresia with/without tracheo-esophageal fistula is a relatively common malformation, Received 24 July 2009 occurring in around 1 in 3500 births. In around half of cases, additional malformations are present, Accepted 4 October 2009 forming either a syndrome of known genetic aetiology, or a recognised association, of which the VAC- Available online 12 October 2009 TERL association (Vertebral anomalies, Anal atresia, Cardiac malformations, Tracheo-Esophageal fistula, Renal and Limb malformations) is the most recognised. Recently, microdeletions of the FOX gene cluster Keywords: at 16q24.1, comprising four genes, FOXF1, MTHFSD, FOXC2 and FOXL1, were reported to cause a phenotype Esophageal atresia resembling VACTERL association, with vertebral anomalies, gastro-intestinal atresias (esophageal, Tracheo-esophageal fistula VACTERL association duodenal and anal), congenital heart malformations, and urinary tract malformations, as well as a rare 16q24.1 microdeletion lethal developmental anomaly of the lung, alveolar capillary dysplasia. This article reviews these new FOXF1 data alongside other genetic causes of syndromic esophageal atresia, and also highlights information Sonic hedgehog from relevant mouse models, particularly those for genes in the Sonic Hedgehog pathway. Ó 2009 Elsevier Masson SAS. All rights reserved.

1. Introduction Genes involved in critical developmental processes, such as tran- scription factors and signalling molecules, often prove to be dosage Esophageal atresia with or without tracheo-esophageal fistula is sensitive. Examples in humans include SOX2 [OMIM 184429]ineye a relatively common malformation, occurring in around 1 in 3500 and foregut development, and N-MYC [OMIM 164840] and, most births [50]. In around half of cases, additional malformations are recently, members of the FOX transcription factor gene cluster at present, forming either a syndrome of known genetic aetiology, of 16q24.1 in foregut and lung development [52]. which CHARGE syndrome [OMIM 214800], The application of array-based comparative genomic hybrid- [OMIM 164280] and AEG syndrome [OMIM 206900] are examples; ization (array-CGH) to the detection of haploinsufficiency in or a recognised association, of which the VACTERL association humans has therefore proved to be very useful for the identifica- [OMIM 192350] is the best recognised. Esophageal atresia and the tion, amongst other things, of developmentally critical, dosage- VACTERL association have been the subject of several recent sensitive, genes. This technique is now fully integrated into clinical reviews [5,16,50]; however, new data concerning the FOX tran- cytogenetics laboratories [2], and in the future may well supersede scription factor gene cluster at 16q24.1 [52] have provided addi- analysis of G-banded metaphase cells by light microscopy. It has the tional insights, making another look at genetic aetiologies of ability to detect copy number loss (and gain) with extremely high esophageal atresia and the VACTERL association worthwhile. resolution, far higher than was previously achievable in a routine A significant factor which has expedited this research is the clinical cytogenetics laboratory only a few years ago. This has had application of high-resolution array-based methods to patients two related, beneficial effects: the first is that more pathogenic with congenital malformations [25,49]. Chromosomal deletions in deletions are being identified; the second is that the deletions are humans result in haploinsufficiency and may therefore disrupt the much smaller, and so contain orders of magnitude fewer candidate normal function of any dosage-sensitive genes which they harbour. genes. Some of the results from the studies using human subjects have been confirmed in other model systems. For example, the efforts of * Tel.: þ44 1223 834244; fax: þ44 1223 494919. mouse developmental biologists have been hugely valuable in E-mail address: [email protected] characterizing gene function in developmental processes by

1769-7212/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.ejmg.2009.10.001 C. Shaw-Smith / European Journal of Medical Genetics 53 (2010) 6–13 7 targeted mutagenesis of candidate genes in mice. In the field of malformations [3,52] but there is no reference to this organ system foregut and lung development, as in many other fields, work on in the literature on Foxf1 and Foxc2 mouse mutants. signalling molecules and transcription factors in a wide range of In the cardiovascular system and gastro-intestinal tract, these pathways is gradually building up a picture of key events in correlations are not as simple. FOXC2 is clearly implicated in morphogenesis of the foregut, lung and other organ systems cardiovascular malformations, albeit with low penetrance: [6,33,44]. This work offers a panel of candidate genes to human in mutation cases [3] and interrupted aortic arch geneticists as they analyze the loci which emerge from array data. in whole cluster deletion cases [52], correlating well with mouse In this review, I focus initially on phenotypes associated with mutants, which have aortic arch malformations [19]. However, one microdeletions at 16q24.1, and the role of the individual genes in of the human FOXF1 mutation cases had a partial AV canal defect the FOX transcription factor cluster. I then go on to place these new [52]; and no such malformations have been reported in Foxf1 data in the context of other single gene disorders associated with mutant mice. syndromic esophageal atresia. The contribution of mouse models to In the GI tract, FOXF1 mutations appear consistently to cause our understanding is discussed, followed by a section focussing on intestinal malrotation, as well as a collection of other GI tract the role of Sonic hedgehog and genes in that pathway. anomalies: duodenal stenosis, congenital short bowel, and annular pancreas, but no gastro-intestinal atresia [52]. This contrasts with heterozygous Foxf1 null mice, which have esophageal atresia and 2. FOXF1 microdeletions at 16q24.1 tracheo-esophageal fistula on selected genetic background (CD1), but in which neither intestinal malrotation, nor indeed any other GI Recently it was shown that microdeletions encompassing the tract anomaly, have been reported [35]. In contrast again, humans FOX cluster at 16q24.1 result in alveolar capillary dysplasia with whole cluster deletions have not only EA/TEF but also together with a broad spectrum of additional malformations [52]. duodenal and anal atresia [52]. No GI tract malformations have Alveolar capillary dysplasia with misalignment of pulmonary veins been reported in humans or mice with FOXC2/Foxc2 mutations. The (ACD/MPV) is a rare lethal disorder associated chiefly with failure possible implications of these observations, and their relationship of development of the intrinsic pulmonary vasculature [[52] and to the Sonic Hedgehog pathway, are discussed further below. references therein]. There is minimal response to respiratory Not only whole cluster deletions, but also deletions upstream of supportive measures and death usually occurs within the first the 16q24.1 FOX cluster, are associated with an abnormal pheno- month of life. In around 80% of cases, additional malformations are type. Such deletions reproduce the alveolar capillary dysplasia present, and the elucidation of the genetic pathology at 16q24.1 in phenotype (cases D9 and D10, Fig. 1) and are also associated, in one a significant proportion of these cases has enabled some inter- case, with anal atresia (case D9, Fig. 1), suggesting that the ‘critical esting genotype–phenotype correlations to be made. Patients with interval’ for GI atresias may not actually include the FOX cluster deletions spanning the entire FOX cluster (FOXF1, MTHFSD, FOXC2 itself (Fig. 1). Future studies will focus on refining this interval by and FOXL1) at 16q24.1 (these are hereinafter referred to as ‘whole the ascertainment of additional deletion cases. cluster deletions’) have, in addition to ACD/MPV, esophageal Mutations in MTHFSD and FOXL1 have not been described in atresia, tracheo-esophageal fistula, and other gastro-intestinal tract humans. Targeted disruption of Foxl1 in mice is associated with atresias (duodenal and anal atresia). Congenital heart defects, hyperplasia of the gastric mucosa, abnormal crypt architecture and urinary tract malformations, vertebral or axial malformations and epithelial cell positioning in the small intestine and retarded are also present, making the resemblance to growth [32,54]; its role in humans will be unclear until mutations the VACTERL association a compelling one. This raises the possi- or single gene deletions of FOXL1 are described in humans. bility that some cases of 16q24.1 microdeletion may be mis- Comparison of the phenotypes of patients with FOXF1, FOXC2 diagnosed as VACTERL association, and the accompanying and whole cluster deletions leads to a hypothesis that the pheno- diagnosis of ACD/MPV overlooked. The diagnosis of ACD/MPV can type in patients with whole cluster deletions results from the be made only by histology, either at post mortem or by examina- contiguous deletion of FOXF1 and FOXC2 with perhaps additional tion of lung biopsy tissue; and even then may be missed, as it is effects from the other genes or elements in the cluster. This model rare and requires some expertise on the part of the pathologist to appears to hold for vertebral, cardiac and renal malformations, but make it. not in the GI tract, where malformations seen in FOXF1 mutation Of the four genes in the 16q24.1 FOX cluster, two, FOXF1 [OMIM cases are different from those seen in whole cluster deletion 601089] and FOXC2 [OMIM 602402] have now been reasonably well patients. This apparent puzzle is discussed further in the section on studied both in humans and the mouse; the remaining two, the Sonic Hedgehog pathway, below. MTHFSD and FOXL1 [OMIM 603252] have been less well studied. Aside from ACD/MPV, there are some phenotypic differences Mutations in FOXC2 cause lymphoedema–distichiasis syndrome between 16q24.1 microdeletion syndrome and VACTERL associa- [OMIM 153400]. A comparison of the phenotypes due to FOXF1 and tion. Hypoplastic left heart syndrome has not previously been FOXC2 mutations in humans, and to Foxf1 and Foxc2 inactivation in reported as a cardiac manifestation of VACTERL association. Limb mouse is shown in Table 1, together with the phenotypes resulting malformations, particularly of the thumb and radius, are a part of from deletions of the whole FOX cluster in humans. For some organ the VACTERL association but do not appear to be present in patients systems, clear interpretation and correlation for the two genes in with 16q24.1 microdeletions, while other malformations not part of human and mouse is possible. For example, in the vertebral/axial VACTERL do occur, such as cleft lip and palate in one patient [52]. system, FOXC2 mutations, whole cluster deletions, and Foxc2 knockout mice all result in vertebral/axial malformations 3. Syndromic esophageal atresia/tracheo-esophageal fistula [3,11,19,52]; neither humans with FOXF1 mutations nor Foxf1 as a single gene disorder knockout mice appear to manifest these. Similarly, FOXF1 and Foxf1, but not FOXC2 or Foxc2, are clearly associated with abnormalities of Four disorders in which EA/TEF feature in a significant number intrinsic pulmonary vasculature and lung lobation [23,28,34] while of cases, and for which the genetic defect has been elucidated, have inactivation of FOXC2 and Foxc2, but not FOXF1 or Foxf1, is associ- been described: these are Feingold syndrome [OMIM 164280], ated with cleft palate [3,19]. Mutations in both FOXF1 and FOXC2,as CHARGE syndrome [OMIM 214800], AEG syndrome [OMIM well as whole cluster deletions, result in urinary tract 206900] and VACTERL-H syndrome [OMIM 276950] (see Table 2 8 C. Shaw-Smith / European Journal of Medical Genetics 53 (2010) 6–13

Table 1 Phenotypes associated with haploinsufficiency of FOXF1 and FOXC2 in humans and of Foxf1, Foxc2 and Shh in mouse.

Gene/ Vertebrae Anal Cardiac Tracheo- Renal Limb Respiratory Cranio-facial Other References locus Esophageal FOXF1 – – AV canal – Hydronephrosis, – ACD/MPV, – Intestinal malrotation, [52] defect, PDA hydroureter, abnormal lung annular pancreas, duodenal dilated bladder lobation stenosis, congenital short bowel, Foxf1 – –– EA/TEF – – ACD-like but no – Gall bladder small with [23,28,35] MPV. Lung abnormal smooth muscle lobation anomalies FOXC2 , rib – Tetralogy of – Duplex , –– Cleft palate Lymphoedema–distichiasis, [3,11] fusion Fallot, VSD, pyelonephritis, varicose veins, ptosis PDA urinary tract infections Foxc2 Split neural – Interrupted –– –– Cleft palate, – [19] arches, absent aortic arch, hypoplastic/ spinous aortic fused middle processes, rib coarctation, ear bones fusions aortic atresia, VSD Deletions Butterfly Anal HLHS, EA/TEF Hydronephrosis, – ACD/MPV, Cleft lip, cleft Single umbilical artery [52] of vertebra, rib atresia Tetralogy of renal pneumothorax palate, whole fusions Fallot, pelvicaliectasis pulmonary brachycephaly FOX Interrupted lymphangiectasia cluster aortic arch, PDA Shh Agenesis of Anal Abnormalities EA/TEF Limb Lung lobation Intestinal malrotation, [9,29,46] axial skeleton atresia of cardiac truncation annular pancreas, duodenal looping defects stenosis, abnormal gut innervation, intestinal transformation of stomach

AV=atrioventricular, VSD=ventricular septal defect, PDA=patent ductus arteriosus, HLHS=hypoplastic left heart syndrome, ACD/MPV=alveolar capillary dysplasia/misalign- ment of pulmonary veins.

and references therein). For the purposes of comparison, the disorders featuring esophageal atresia for which there is as yet no 16q24.1 locus is added to this list in Table 2, accepting the fact that confirmed genetic aetiology include [OMIM the genetic mechanism in this disorder has not been fully eluci- 229850] [1], and congenital microgastria–limb reduction complex dated, and that it may be a contiguous gene deletion syndrome. [OMIM 156810] [31], discussed further below. Finally, there are There are some striking similarities between the five disorders, several reports of a syndrome of multiple gastro-intestinal atresias, as well as some differences. Vertebral and urinary tract malfor- including esophageal atresia/tracheo-esophageal fistula, with gall mations occur in all five. All except AEG syndrome feature cardio- bladder agenesis and neonatal mellitus, associated in vascular malformations, with aortic arch abnormalities featuring some but not all cases with pancreatic anomalies [8]. Parental prominently, and likewise, anal atresia or stenosis occur in these consanguinity and sibling recurrence point to autosomal recessive four. GI tract malformations are very similar in Feingold syndrome inheritance, but there are as yet no mapping data. and 16q24.1 microdeletions, with EA/TEF, , anal atresia, and annular pancreas occurring in both. There is least 4. Mouse models featuring esophageal concordance for limb malformations, which are of different types in atresia/tracheo-esophageal fistula the three conditions in which they occur, VACTERL-H, Feingold and Charge syndromes. There is clearly a danger in overstating the A list of mouse models featuring esophageal atresia or tracheo- similarities between these diverse conditions, especially as the esophageal fistula is provided in Table 3. Prominent on this list are column labelled ‘other’ contains many anomalies not otherwise members of the Sonic Hedgehog pathway, including Sonic classified in the table, but nonetheless, similarities both across and Hedgehog itself. Shh/ mice have an extensive set of malforma- within organ systems, such as those between Feingold syndrome tions which overlaps very significantly not only with the VACTERL and 16q24.1 microdeletion syndrome, are striking and hint at association, but also with the set of malformations seen in patients similarities in pathogenesis. with microdeletions at 16q24.1 (excluding ACD/MPV) [9,52]. Esophageal atresia/tracheo-esophageal fistula occur occasion- Mutations in SHH in humans cause [HPE3, ally in other single gene disorders. These include Opitz B/GGG OMIM 142945] [40] and microphthalmia [OMIM 611638] [48]. syndrome [OMIM 300000], due to mutations in MID1 [OMIM Mutations have not been reported in patients with malformations 300552] at Xp22.3 [42], in which laryngeal malformations (cleft, of the gastro-intestinal tract. The GLI family of transcription factors diastema) are more usual; McKusick–Kaufman syndrome [OMIM are downstream effectors of SHH and in humans, mutations in 236700], which is allelic with Bardet–Biedl syndrome [BBS-6, members of this gene family result in syndromes well known to OMIM 209900] and due to mutations in MKKS [OMIM 604896] clinical geneticists. Greig cephalopolysyndactyly [GCPS, OMIM [51,53], a gene related to members of the chaperonin family; and 175700] and Pallister–Hall [PHS, OMIM 146510] syndromes are Oculo-Auriculo-Vertebral Spectrum [OAVS, OMIM 164210], for caused by mutations in GLI3 [20], while mutations in GLI2 are which epigenetic dysregulation of BPAX1 has been proposed as an associated with holoprosencephaly [47]. None of these syndromes aetiological mechanism [14]. Putative single gene or sporadic manifests foregut malformations, although anal atresia is C. Shaw-Smith / European Journal of Medical Genetics 53 (2010) 6–13 9 FLJ30679 FOXL1 FOXF1 FOXC2 MTHFSD LOC732275 COX4NB USP10 KIA182 C16ofr74 MAP1LC3B CRISPLD2 KIAA0513 GINS2 COX4i1 FBX031 ZCCHC14 JPH3 IRF8 KIAA1609 COTL1 ATP2C2

83 84 85 86 D1 Mb from 16pter D2 D3 D4 D5 D6 D7 D8 D9 D10 FLJ30679 FOXF1 FOXL1 FOXC2 MTHFSD LOC732275 C16ofr74 COX4NB KIA182 MAP1LC3B COTL1 USP10 KIAA1609 CRISPLD2 KIAA0513 GINS2 COX4i1 FBX031 JPH3 IRF8 ZCCHC14 ATP2C2

38 48 58 86 Mb from 16pter D1 D2 D3 D4 D5 D8 D9 D10

Fig. 1. Microdeletions at 16q24.1 in patients with gastro-intestinal atresias Three patients in the recent report of microdeletions at 16q24.1 presented with gastro-intestinal atresias: D1, D3 and D9. One of these, D9, is a deletion upstream of the FOX transcription factor cluster, suggesting a ‘critical interval’ for gastro-intestinal atresias at this locus, that does not span the FOX cluster itself. More deletion cases are needed in order to confirm or refute these preliminary ideas.

a component of Pallister–Hall syndrome; however, mice with 5. Dissection of Shh and Foxf1 function in model organisms inactivation of both Gli2 and Gli3 do have foregut malformations (summarized in Table 3), and again it seems worthwhile to search Key emerging players in the pathogenesis of esophageal atresia, for mutations in GLI family members in patients with syndromic tracheo-esophageal fistula and the VACTERL association are esophageal atresia. members of the Sonic Hedgehog pathway, and of the FOX tran- Mice with Noggin/ mutations have a wide range of malfor- scription factor gene cluster at 16q24.1. Studies in model organisms mations including esophageal atresia and tracheo-esophageal including Xenopus and mouse have demonstrated key roles for fistula [27,43]. Tantalizingly, a clear link between the NOGGIN locus Sonic Hedgehog itself, and for Foxf1, in mediating mesoderm– at 17q22 and esophageal atresia has been established in humans endoderm cross talk during very early development of the gut tube. [36], with most recently, the critical interval narrowed to a 5.9 Mb A critical early step is the subdivision of lateral plate mesoderm into region encompassing NOGGIN [41]. The phenotype in these dele- its somatic and visceral components, and evidence from Xenopus tion cases has been tentatively delineated to include deafness and [56] and mouse [34] assigns a key role to Foxf1 in this process. skeletal malformations (symphalangism, joint contractures) as well Subsequently, the dorsal mesentery, from which the early gut tube as EA/TEF, and so mutations in NOGGIN could usefully be sought in is suspended from the dorsal abdominal wall, and the splanchnic deletion-negative patients with this phenotype. mesoderm lining the gut tube, develop from the visceral compo- Finally, a mouse model was published recently in which muta- nent of the lateral plate mesoderm. Signalling between the visceral tion of the proprotein convertase enzyme Pcsk5 was shown to be mesoderm, which develops into smooth muscle and other associated with malformations in the VACTERL spectrum, including specialized tissues in the gut wall, and the gut endoderm, which defects of tracheo-oesophageal septation [60]. Convincing muta- provides the epithelial cell lining of the gastro-intestinal tract, is tions in this gene in humans with VACTERL association have yet to critical for correct development of the gut [37]. There is compelling be identified. evidence that Foxf1 is a downstream effector of the Sonic hedgehog 10

Table 2 Syndromic esophageal atresia due to single gene disorders in humans, with microdeletion at 16q24.1 for comparison.

Syndrome/gene Vertebrae Anal atresia/other Cardiac Tracheo- Renal Limb Other References GI malformations Esophageal

Feingold syndrome Fused cervical Anal atresia, also Tricuspid EA, TEF Hydronephrosis, Short middle , learning [7] N-MYC vertebrae, absent duodenal atresia stenosis/atresia, cystic dysplasia, phalanges of 2nd difficulties, deafness, short

sacral vertebrae, 6–13 (2010) 53 Genetics Medical of Journal European / Shaw-Smith C. or stenosis, interrupted aortic dilatation of renal and 5th digits, absent rib pairs stature, asplenia or polysplenia annular pancreas arch, VSD, PDA , small kidneys hypoplastic thumbs, toe (2nd and 3rd, or 4th and 5th toes)

VACTERL- Lumbar spina bifida Anal atresia Aortic coarctation, EA, TEF Unilateral renal Bilateral radial agenesis, Fanconi anaemia, , [18,30,38] hydrocephalus occulta, cervical ASD, VSD, agenesis, renal absent thumbs Arnold–; cleft FANCB vertebral defects dysplasia palate, incomplete lung lobation

Charge syndrome Vertebral body Anal stenosis HLHS, Tetralogy of EA, TEF Renal agenesis, Monodactyly, tibial Coloboma of eye, structural anomalies [21,57] CHD7 anomalies, Fallot, DORV, AVSD, horseshoe kidney, aplasia, bifid femora of external ear, deafness, agenesis of kyphoscoliosis right-sided descending vesico-ureteric reflux, semi-circular canals, choanal atresia, aorta, Shone’s complex, renal cysts cleft lip, cleft palate, cryptorchidism, ASD, VSD, PDA micropenis, hydrocephalus, corpus callosum agenesis, seizures, learning difficulties

AEG syndrome Hemi-vertebrae, – – EA, TEF Hypoplastic kidneys, – Anophthalmia, microphthalmia, [59] SOX2 butterfly vertebrae, duplex kidneys hypospadias, cryptorchidism fused ribs, absent ribs, extra ribs

16q24.1 Butterfly vertebra, rib Anal atresia, HLHS, Tetralogy of Fallot, EA, TEF Hydronephrosis, – ACD/MPV, cleft lip, cleft palate, [52] microdeletion fusions posterior placement Interrupted aortic arch, renal pelvicaliectasis brachycephaly, single umbilical artery encompassing of anus, duodenal PDA FOXF1, MTHFSD , atresia, annular FOXC2 and FOXL1 pancreas

AVSD=atrioventricular septal defect, ASD=, VSD=ventricular septal defect, PDA=patent ductus arteriosus, HLHS=hypoplastic left heart syndrome, ACD/MPV=alveolar capillary dysplasia/misalignment of pulmonary veins, DORV=double outlet right ventricle. C. Shaw-Smith / European Journal of Medical Genetics 53 (2010) 6–13 11

Table 3 Knockout mouse models featuring esophageal atresia and/or tracheo-esophageal fistula, with phenotype associated with inactivation of counterpart human gene for comparison.

Mouse Lung/foregut phenotype Phenotype (other) Reference Human Locus Mutations Lung/ Phenotype (other) Reference gene gene foregut phenotype Shh Esophageal atresia, tracheo- Abnormalities of CNS; cyclopia; [9,29,46] SHH 7q36 YES NR Holoprosencephaly [HPE3, [40,48] esophageal fistula, lung distal limb truncation; OMIM 142945], anomalies abnormalities of axial skeleton, microphthalmia [OMIM renal agenesis, abnormalities of 611638] heart looping, intestinal malrotation, annular pancreas, duodenal stenosis, intestinal transformation of the stomach, abnormal gut innervation,

Foxf1 Esophageal atresia, tracheo- Axial skeletal defects [22,35] FOXF1 16q24.1 YES Alveolar Intestinal malrotation, atrio- [52] esophageal fistula, lung lobe capillary ventricular canal defect, renal fusion defects, pulmonary dysplasia malformations vascular defects

Gli2 Gli2/ mice are normal. Gli2 NR [39] GLI2 2q14 YES NR Holoprosencephaly [47] / Gli3þ/ mice have esophageal atresia tracheo- esophageal fistula. Gli2/ Gli3 / mice have absent oesophagus, and lungs

Gli3 See Gli2 entry Gli3/ mice are allelic with Xt. [39] GLI3 7p13 YES NR Greig cephalopolysyndactyly [20] They have and syndrome [GCPS, OMIM brain malformations. 175700]; Pallister–Hall syndrome [PHS, OMIM 146510] (imperforate anus, , hypopituitarism, hypothalamic hamartoblastoma)

Noggin Esophageal atresia, tracheo- Failure of closure of neural tube, [27,43] NOGGIN 17q22 YES NR Proximal symphalangism with [4,17,26] esophageal fistula exencephaly, wide, club-shaped multiple synostoses; stapes limbs, shortened, abnormal ankylosis with broad thumbs body axis, lethality at birth and toes; brachydactyly type B2

Sox2 Esophageal atresia, tracheo- Neurodegeneration, impaired [13,44] SOX2 3q26.3 YES Esophageal Anophthalmia, genitourinary [59] esophageal fistula neurogenesis atresia malformations

NR ¼ not recorded. pathway in this process, both in the lung and in the gut. In the lung, to VACTERL association have already been commented upon. How ectopic epithelial expression of Shh activates Foxf1 expression [35]; do we relate these observations from model organisms to relevant in Shh/ mice, Foxf1 mRNA is absent from its usual sites of human phenotypes? For SHH, human mutations so far described expression in mesenchyme of trachea and oesophagus, and lungs, have resulted in holoprosencephaly and microphthalmia, but these although midgut and hindgut do retain some expression [35]. have so far been mis-sense mutations or in-frame deletions. Where Similarly, in the intestine, but not the stomach, of both Gli2 and Gli3 are patients with inactivating mutations and deletions of this gene? null mice, Foxf1 levels are reduced [32]. The Foxf1 promoter, as well One possibility is that these mutations are lethal at an early as that of Foxl1, contains binding sites for Gli factors which mediate embryonic stage; a second possibility is that investigators have not transcriptional activation [32]. It does appear that expression of looked in the right phenotypic group. One reported phenotype is the Foxf1 is not exclusively controlled by the Sonic Hedgehog pathway, Microgastria–Limb Reduction complex [OMIM 156810], which as evidenced by its retained expression in the intestine of Shh/ features terminal transverse limb defects similar to those seen in mice [35], but clearly there is some regulation of Foxf1 by this Shh/ mice, intestinal malrotation, esophageal and anal atresia, pathway, and the close link between the two is evident in the megacolon, abnormal lung lobation, heart defects, renal and CNS phenotypic similarities of mouse mutants and human patients with anomalies. This shows some similarities to the phenotype of Shh/ FOXF1 mutations and deletions [52]. More detailed analysis of GI mutant mice, but, against this, three reported cases of this condition tract malformations in Shh/ mice has revealed more extensive have been in discordant twin pairs, suggesting that the aetiology abnormalities: intestinal malrotation, annular pancreas, duodenal may be related to the twinning process [31]. stenosis, abnormal gut innervation, and intestinal transformation Concerning FOXF1 and the 16q24.1 FOX gene cluster, the inter- of stomach [46]. Strikingly, the first three malformations on this list esting question remains: why does FOXF1 inactivation result in are also seen in patients with FOXF1 mutations [52]. Sonic Hedgehog intestinal malrotation, but deletion of the entire FOX cluster result therefore appears to provide a link between the set of malforma- in gastro-intestinal atresias, whereas Foxf1 inactivation in mice tions seen in FOXF1 mutation cases and the set seen in whole results in esophageal atresia? Future studies will focus on nar- cluster deletion cases: the malformations that occur are all seen in rowing the ‘critical interval’ responsible for occurrence of GI atre- Shh/ mice. The relationship between Shh and Foxf1 is further sias (esophageal, duodenal and anal) in patients with explored in Fig. 2. microdeletions at 16q24.1. It will be interesting to determine In mice, the phenotypic consequences of Shh, Gli transcription whether FOXL1, which also has a role in intestinal development, factor and Foxf1 inactivation are shown in Table 3; the resemblances contributes to this phenotype in humans. 12 C. Shaw-Smith / European Journal of Medical Genetics 53 (2010) 6–13

further genotype–phenotype studies are required in order to clarify precisely which genes or sequence elements are responsible.

7. Concluding remarks

The recent delineation of the role of the 16q24.1 locus in the aetiology of severe developmental malformations illustrates very clearly the role that high-resolution microarrays can play in improving our understanding of developmental disorders. All of the deletions reported in affected patients are below the level of cyto- genetic resolution, but very easily detectable using microarrays. Recent work on chromosomal anomalies in EA/TEF suggests that there are other haploinsufficient susceptibility loci for these and related malformations, notably at 13q and 17q, and quite naturally as a result of clinical studies, our knowledge will grow. There is also a clear utility in research studies focussed on a particular malfor- mations: our own study, the Genetics of Oesophageal Atresia http:// www.ich.ucl.ac.uk/ich/academicunits/MMU/CustomMenu_01 now has high-resolution array data on close to 100 patients with syn- dromic esophageal atresia, with some potentially interesting loci. These data will be published in due course. A complementary approach is the sequencing of genes in the same cohort of patients, looking for mutations both in known (N-MYC, SOX2 and others) and in candidate (SHH, NOG and others) genes. Sequencing can be carried out on a large scale on whole- genome amplified DNA, as has recently been described for the whole X chromosome [55]. Ultimately, the goal of these studies is to complete the ‘cytogenetic map’ for esophageal atresia and tracheo- esophageal fistula. This work is now well under way, and will be useful not only for its own sake but also as a complement to studies of non-genetic factors in EA/TEF, which have to date been restricted mostly to large-scale epidemiological studies. Fig. 2. Epithelial–mesenchymal interactions mediated by the Sonic Hedgehog pathway in the gut. Sonic hedgehog effects signalling between the epithelial and Acknowledgements mesenchymal cell in the gastro-intestinal tract, and acts indirectly upon Foxf1 via its receptors, Smo and Ptch, and Gli transcription factors. The effects of Foxf1 are mediated C.S.-S. is the recipient of a Wellcome Trust Intermediate Clinical within the mesenchyme, but there is currently much uncertainty surrounding the identity of the downstream effectors of Foxf1. Shh, Sonic Hedgehog; Ptch, Patched; Fellowship, and gratefully acknowledges additional funding from Smo, Smoothened. the Addenbrooke’s Charitable Trust and Tracheo-Oesophageal Fistula Support (TOFS), the UK patient support group for patients 6. Chromosomal imbalances with esophageal atresia and tracheo-esophageal fistula. Thanks to Vicki Martin for technical assistance and to Jianwen Que for A comprehensive review of chromosomal anomalies in esoph- detailed suggestions on the manuscript. ageal atresia/tracheo-esophageal fistula was published recently [12], and in this section, attention is drawn to just a few of these. References Trisomies for chromosomes 18 and 21 have been associated with EA/TEF; interestingly more cases have been associated with trisomy [1] S. Ayme, C. Julian, D. Gambarelli, B. Mariotti, A. Luciani, N. Sudan, et al., Fryns 18 despite the fact that it is much rarer than trisomy 21 [50]. The syndrome: report on 8 new cases. Clin. Genet. 35 (1989) 191–201. locus perhaps closest to revealing a new causative gene is 17q22, [2] A.L. Beaudet, J.W. Belmont, Array-based DNA diagnostics: let the revolution begin. Annu. Rev. Med. 59 (2008) 113–129. harbouring NOG, discussed above [36]. A link between chromo- [3] G. Brice, S. Mansour, R. Bell, J.R. Collin, A.H. Child, A.F. 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