2 Genetics of Anorectal Malformations

Giuseppe Martucciello

Contents blastogenesis often involve two or more progenitor fields. This fact may explain the cause of subgroups 2.1 Introduction . . . 17 of ARM that form part of complex phenotypes due 2.2 Non-syndromic ARM . . . 17 to developmental field defects. These complex- phe 2.3 ARM Associated with Other Systemic notypes are considered as end results of pleiotropic Malformations . . . 18 effects of single causal events that might be chromo- 2.4 Aetiological Classification . . . 20 somal, monogenic or even teratogenic [72]. 2.4.1 Chromosomal Anomalies Classifying ARM from the genetic point of view is Associated with ARM . . . 20 2.4.2 Down Syndrome . . . 20 not easy since they present different forms that are be- 2.4.3 Cat-Eye Syndrome . . . 20 lieved to be influenced by different factors such as sex 2.4.4 Genetic Syndromes Associated and associated anomalies. ARM can be the only path- with ARM . . . 21 ological finding (non-syndromic) or as part of a more 2.4.4.1 Townes-Brocks Syndrome . . . 21 complex phenotype (syndromic), and may occur in a 2.4.4.2 FG Syndrome . . . 21 single affected individual (sporadic) or in more than 2.4.4.3 Pallister-Hall Syndrome . . . 21 one individual in the same family (familial) with dif- 2.4.4.4 VACTERL Association (VATER) . . . 22 ferent modes of inheritance. There are gender differ- 2.4.4.5 Sirenomelia . . . 22 ences, with remarkably higher preponderance in boys 2.4.4.6 Caudal Regression Syndrome . . . 23 for more complex ARM forms, while the less severe 2.4.4.7 Currarino Syndrome . . . 23 types, with perineal or vestibular fistulae, reported 2.5 Mouse Models with ARM . . . 25 more frequently in girls (Fig. 2.1) [61]. These topics 2.6 Conclusions . . . 26 will be discussed in detail below. References . . . 26

2.2 Non-syndromic ARM

Sporadic ARM is a frequent clinical finding; however, 2.1 Introduction different modes of inheritance have been reported. Eleven families have been reported with inherited Anorectal malformations (ARM) present an incidence variants, 7 of these families showing autosomal domi- rate ranging from 1:1,500 to 1:5,000 live births [18, nant mode of inheritance. In one family the condition 19, 41, 49, 52, 53, 61, 109, 110, 112], and have vari- was suggestive of autosomal recessive inheritance, able clinical presentations ranging from mild forms with two affected females to healthy-looking parents. that might require only minor surgical interventions In the remaining three families, it was not possible to to more complicated cases that need to be managed differentiate whether the inheritance was autosomal with multi-staged operations [51, 87, 88, 108]. or X-linked recessive (Table 2.1). The cause of ARM is unknown, although arrest Moreover, excluding the isolated autosomal domi- of the descent of the urorectal septum towards the nant low type, ARM can escape diagnosis easily due cloacal membrane between the 4th and 8th weeks of to variable expression, such as presenting with a very gestation was previously considered the basic event mild manifestation such as an anteriorly located anus leading to ARM [31, 32, 55, 71]. Since the molecu- (perineal fistula) [60]. Careful examination of the first- lar determinants during blastogenesis are overlap- degree relatives, particularly the parents, is highly ping for many body systems, and these elements are recommended before calculating the recurrence risk closely related in timing and spacing, so defects in in isolated low ARM due to variable expression [96]. 18 Giuseppe Martucciello

Fig. 2.1 Classification of anorectal malformations (ARM)

Table 2.1 Suggested mode of inheritance for isolated anorectal malformations (ARM) Reports Affected members Suggested inheritance Manny et al. (1973) [68] Father-son Autosomal-dominant Schwoebel et al. (1984) [106] Mother, son and daughter Autosomal-dominant Boocock and Donnai (1987) [12] Three father-son pairs Autosomal-dominant Weinstein (1965) [124] Three families with affected Autosomal- or X-linked recessive sons and healthy parents Winkler and Weinstein (1970) [126] Two daughters, healthy parents Autosomal-recessive Landau et al. (1997) [60] Four members in three generations Autosomal-dominant Robb and Teebi (1998) [100] Father-son Autosomal-dominant

2.3 ARM Associated with Other affected with ARM of whom 58% had additional Systemic Malformations malformations: 68% were male and 32% were female. High-type ARM was found in 58%, but occurred five There is a wide spectrum of possible associations times more frequently in males than in females, while of ARM with various systemic malformations (Ta- low-type ARM occurred twice as often in females ble 2.2). These associations may be well-described than in males. High-type ARM were associated 13 syndromes with various aetiologies or may be just a times more frequently with other congenital malfor- random association with other malformations. In a mations than were low-type ARM. ARM with other recent review, Ratan et al. [94] studied 416 patients anomalies were four times more common among 2 Genetics of Anorectal Malformations 19 males than females. These findings are in contrast (HD), is another interesting field, since several caus- with isolated ARM, for which there is no sex differ- ative genes for HD are known. The incidence of this ence and, more surprisingly, the isolated low-type combination varies widely from one study to another, ARM is twice as common as the high type. Ratan et al. ranging from 3.4% [54] to 60% [86]. Lerone et al. sug- [94] also found that boys with high ARM had more gested that this variability depends on whether suit- vertebral and gastrointestinal tract anomalies, espe- able techniques for diagnosing intestinal innervation cially for congenital short megacolon, while the boys were used or not [61]. Enzymohistochemical tech- with low ARM had a higher incidence of genital mal- niques studying cryostat sections from biopsy speci- formations. On the other hand, girls with high ARM mens of the fistula and rectal pouch demonstrated an have more skeletal anomalies (other than vertebral) abnormal innervation in 96% of ARM patients [61, and urinary tract malformations. Girls affected with 44]. Classical aganglionosis was found in 31% of rec- low ARM frequently suffered from vesicoureteric re- tal pouch specimens, hypoganglionosis in 38%, and flux. The percentages of patients affected by ARM and intestinal neuronal dysplasia type B in 14%; unclas- 2,3, or 4 other body system anomalies were 50%, 29%, sified dysganglionosis was found in 10%. These data and 16% respectively. demonstrate that ARM and dysganglionoses are not The analysis of associated malformations plays an uncommon, and suggest the importance of mutation important role in the genetic study of ARM, because analysis of HD genes in ARM-affected individuals diagnosing the associated anomalies can identify [61]. specific genes that may represent a starting point for more detailed investigations. Studies have thus fo- cussed on the most frequently associated anomalies, 2.4 Aetiological Classification namely the vertebral, genitourinary and gastrointestinal malformations [61]. The frequent association of ARM with common chro- A condition that is commonly associated with mosomal anomalies is well known (e.g. Down syn- ARM is hypospadias (found in 6.5% of cases) [40]. drome, trisomy 18, 13q-, cat-eye syndrome (CEM), or Moreover, one out of five cases of congenital urethral genetic syndromes such as Currarino syndrome, FG fistula – a rare condition – is associated with ARM. In syndrome, VATER association and others [14, 17, 22, a study on rectal innervation in patients with ARM, 35, 39, 42, 48, 75, 80, 82, 85, 95, 98, 99, 111, 115, 123]. enteric nervous system anomalies ranged from 79 to Teratogenic effects of certain agents such as thalido- 100% of cases depending on the ARM type [61]. mide, oestrogen and ethanol intake has also been well The combination of both ARM and intestinal dys- described [8, 61, 89]. ganglionoses, particularly Hirschsprung’s disease

Table 2.2 Systemic malformations associated with ARM. US Ultrasound, IVU intravenous urethrogram, MCU micturating cysto- urethrogram, MRI magnetic resonance imaging, ECG electrocardiogram Anomalies Incidence in literature (%) Investigations Notes Urologic 25–55% US, IVU Hoekstra, Ratan, Mittal Vesicoureteric reflux 20–47% US, MCU Hassink, Narasimharao, Rickwood, Ratan Genital 3–18% (boys) Hoekstra, Metts, Cortes 26–39% (girls) Vertebral 25–38% X-ray, MRI Hassink, Carson, Mittal, Ratan Other skeletal 13–16% X-ray Hassink, Ratan, Mittal Spinal-cord-related 8–67% X-ray, MRI Rivosecchi, Walton, Ratan, Mittal Cardiac 10–17% ECG, echocardiography Mittal, Greenwood, Ratan Gastrointestinal 7–10% X-ray, biopsy Ratan, Mittal, Hassink Tracheoesophageal 6–8% Hassink, Ratan Others 4–5% Ratan 20 Giuseppe Martucciello

2.4.1 Chromosomal Anomalies Associated 2.4.3 Cat-Eye Syndrome with ARM CES is characterised by ARM, coloboma of the iris In a study of 1,846 babies with ARM [24], chromo- (total or rarely partial, unilateral or bilateral), colo- somal anomalies were found in 11%, the most fre- boma of the choroid and/or optic nerve, microph- quent form being perineal fistula. The frequencies of thalmia (usually unilateral) and variable external ear trisomy 21, 13, and 18 among babies with ARM were deformities ranging from unilateral auricular reduc- 15, 30, and 90 times higher, respectively, in compari- tion defects to several tags, mostly with atresia of the son to neonates in the general population (Table 2.3). external auditory canal. These anomalies can be associated with mental retardation in half of the cases; dysmorphic features are the leading signs for the di- 2.4.2 Down Syndrome agnosis, including hypertelorism, downward-slanting palpebral fissures and low root of the nose. Con- The ARM occurs more frequently with Down - syn genital heart disease can also be associated with this drome than in the general population and there is a syndrome, especially septal defects and anomalous higher rate of deformities without fistula [11, 116]. pulmonary venous return, as can different forms of In a study of 1,992 patients with ARM in Japan [30], urinary tract malformations such as renal agenesis or Down syndrome was seen in 101 patients (5.1%), hypoplasia, vesicoureteral reflux and bladder anoma- with no gender variation. The incidence of high, in- lies [7, 61, 105]. termediate, and low types were 2.7%, 18.7% and 4.1%, CES is usually associated with the cytogenetic respectively, showing clearly the statistical difference finding of a supernumerary marker chromosome between the intermediate type and the others. Ninety- consisting of duplicated material of chromosome 22. five percent (96 out of 101) of patients with Down Bisatellite and dicentric markers are usually found syndrome had deformities without fistula, while only (idic(22)(pter→q11.2::q11.2→pter)) and thus results 3 patients had rectourethral fistula; the remaining 2 in tetrasomy of the p arm and a part of 22q11.2 [74, had a perineal fistula. 105]. This chromosomal anomaly generally follows a de novo mutation, and the recurrence risk does not increase with subsequent pregnancies. However, in a few cases the anomaly may segregate from an affected parent. Mosaicism can be a frequent finding in blood samples of the affected index case and of his family members. This may draw attention to considerable Table 2.3 Some forms of chromosomal anomalies associated intrafamilial variability of the phenotypic expression, with ARM (from Cuscheri et al. 2002) [24] and the potential recurrence risk for patients with a Chromosomal anomalies Number of cases out of normal karyotype [105]. 1,846 ARM patients Trisomy 8 mosaic 1 2.4.4 Genetic Syndromes Associated Trisomy 13 12 with ARM Trisomy18 20 Trisomy 21 39 Seven major syndromes have been considered in this Trisomy 22 3 chapter: Townes-Brocks syndrome (TBS), FG syndrome, Pallister-Hall syndrome (PHS), VACTERL Sex chromosome aneuploidy 4 (vertebral, anorectal, cardiac, tracheoesophageal, re- Triploidy 1 nal, and limb anomalies) Association (VATER), sire- Tetrasomy 12 p (Pallister-Killian) 2 nomelia, caudal regression syndrome (CRS) and Cur- Ring (13) 3 rarino syndrome. Deletion 5p 3 Extra fragment 3 2.4.4.1 Townes-Brocks Syndrome Partial tri/monosomy 21 Other chromosome anomalies 17 Otherwise known as renal-ear-anal-radial syndrome or Townes-Brocks-branchio-oto-renal-like syndrome, 2 Genetics of Anorectal Malformations 21 this autosomal dominant syndrome was first -de 2.4.4.2 FG Syndrome scribed in 1972 [116]. It is estimated to have an incidence rate of 1:250,000 live births [70]. TBS has great FG syndrome, which takes its name from the initials variability among affected families [89], but in gen- of the first described case [83], is characterised by eral the main characteristic features of TBS are: (1) mental retardation, and multiple congenital anoma- ARM (imperforate anus and anal stenosis), (2) hand lies including large head, imperforate anus, congeni- malformations in the form of preaxial polydactyly tal hypotonia and partial agenesis of corpus callo- with diverse thumb anomalies ranging from vestigial, sum). It can involve many body systems, including: broad, to triphalangeal thumb, and even distal ulnar (1) the central nervous system (most markedly men- deviation of the thumb and (3) external ear malfor- tal retardation, congenital hypotonia, convulsions, mation (microtia, external auditory atresia, satyr ear and sensorineural deafness) – the malformations that with overfolding of the superior helix, preauricular can be seen through imaging techniques are partial pits and sensorineural deafness). Other associated or total agenesis of the corpus callosum, hydrocepha- anomalies are congenital heart malformations, mostly lus, megaloencephaly and neuronal migration defect; tetralogy of Fallot, ventricular septal defect, truncus (2) dysmorphic features: frontal bossing, macroceph- arteriosus and genitourinary anomalies (which can aly, hypertelorism, telecanthus, epicanthal folds and include dysplastic kidneys, vesicoureteric reflux and downward-slanting palpebral fissures; (3) severe con- hypospadias); mental retardation has been noted in tipation that can be associated with ARM; (4) genital variable degrees [89]. The major criteria for the diag- malformations: cryptorchidism, hypospadias, hernia; nosis of TBS are the hands, ears and anus. In the pres- (5) patients usually have fine, silky, and soft hair with ence of only two major criteria the diagnosis becomes an anterior upswept hairline and excessive number less secure and may be taken in consideration only of hairwhorls; (6) ocular abnormalites in the form if: (1) they are accompanied by minor malformations of squint and ptosis; (7) broad thumbs and halluces. such as cardiac and renal anomalies and deafness, (2) This syndrome is X-linked recessive with a gene map absence of atypical features (i.e. tracheoesophageal or locus on Xq12-q21.31. vertebral anomalies), (3) presence of another affected individual in the family and (4) other affected per- sons in the family who have the missing major feature 2.4.4.3 Pallister-Hall Syndrome in the index case. In that case the clinical diagnosis can be confirmed by a mutational analysis of the This is a rare, life-threatening disorder that is char- causative gene SALL1. Failure to detect the mutation acterised by hypothalamic hamartoma (commonly does not rule out the diagnosis since the detection leading to precocious puberty or panhypopituita- rate is 64.3–83.3% of patients with “classical” TBS rism), polydactyly (central), imperforate anus, and with hands, ears and anal malformations. SALL1 gene respiratory tract anomalies (bifid epiglottis and/or is the only known gene causing TBS [56, 58, 69]. TBS other laryngeal anomalies) [37]. The mode of inheri- exhibits similar features to other syndromes, namely tance is autosomal dominant, with remarkably vari- Goldenhaar, VACTERL or oculo-auriculo-vertebral able expression [10, 102]. PHS (together with Greig [89]. SALL1 was mapped first to 16q12.1 by fluores- cephalopolysyndactyly), as a distinct and pleiotropic cence in situ hybridisation [57]. Up to 29 mutations developmental anomaly, is caused by mutations in were reported in affected individuals with scattered the gene GLI3, which is inherited in an autosomal mutations all over the gene [56]. However, it is still dominant pattern [50, 121]. unknown whether certain forms of mutations in PHS disease tends to be expressed mildly in fa- other parts of SALL1 would result in different pheno- milial cases, while it takes a more dramatic course in types. Mutation analysis of SALL1 has confirmed that sporadic cases. The estimate of recurrence risk and penetrance is complete in TBS [56]. Prenatal diagno- genetic counselling should be based on whether the sis can be performed by searching for mutations of index case is part of a familial condition. In this case the SALL1 gene in amniotic fluid and chorionic villus the disease usually tends to repeat itself in successive samples. In the case of absence of known mutations generations of affected individuals, with a 50% recur- of SALL1 in affected families, prenatal diagnosis can rence risk. Incomplete penetrance is not reported and be performed by high-resolution ultrasound between those who harbour the mutation usually manifest the 18 and 22 weeks of gestation, by finding the thumb disease, but with highly variable expression. However malformations since ARM can not be seen easily, it is important to inform these families about the pos- while renal anomalies can be identified [56]. sibility of having a baby affected with PHS in a more 22 Giuseppe Martucciello

severe or even milder form than that of the other in- TERL, these can be trisomy 18 or long arm deletion dividuals in the family. Regarding sporadic cases, in of chromosome 13 [1, 6, 43, 110]. Recently, a case of whom the disease tends to occur in a severe form and VATER with 9q+ was reported [2], similarly a case of frequently with a higher mortality rate and reduced interstitial deletion of the long arm of chromosome 6: reproductive fitness, the recurrence risk can be that of del (6) (q13q15) in association with VACTERL asso- the general population. However, the absence of cases ciation [122], and another male case with VACTERL with gonadal or germinal mosaicism does not mean association and a karyotype showing mosaicism for that it can not exist, so the family should be informed a supernumerary ring chromosome in 63% of all the about the substantial risk of recurrence [9]. metaphases of both the lymphocytes and fibroblasts. This ring chromosome belongs to chromosome 12 [20]. A patient affected with VACTERL has also been 2.4.4.4 VACTERL Association (VATER) found to harbour a somatic point mutation in mitochondrial DNA obtained from kidney tissue [25]. VACTERL is an acronym for vertebral anomalies (fusion, hypoplasia), ARM, cardiac malformations, tracheoesophageal fistula with or without atresia, renal 2.4.4.5 Sirenomelia anomalies (renal agenesis, hypoplasia or even cystic dysplasia) and limb anomalies (usually involving Sirenomelia may represent one of the oldest diag- the radial ray such as radial or thumb hypoplasia, nosed congenital deformities, since it was mentioned either uni- or bilateral). This term represents the ex- by the ancient Greeks. Sirenomelia manifests with panded previous acronym of VATER, which stands fusion of the lower limbs at a varying level and de- for vertebral defects, anal atresia, tracheoesophageal gree, with inability to perform normal movements fistula, and radial dysplasia, first reported by Quan and rotation (apodia, monopodia and dipodia). Not and Smith [91]. VACTERL is believed to result from only are limb anomalies found in this lethal phenom- an early embryonic insult, more specifically of blas- enon, but also ARM (variable forms, including cloa- togenic origin occurring during the first 4 weeks of cal anomalies), genital (absent or arrested develop- embryogenesis, so the expected effects are primary, ment), renal (cystic kidneys, or agenesis as a common polytopic, developmental field defects [70]. This cause of death when bilateral), gastrointestinal tract early embryonic event can lead to different defects in malformation, skeletal (vertebral and rib anomalies), various body systems. Of the 416 patients with ARM various upper-limb defects, congenital heart disease described by Ratan et al. [93], the additional anoma- and more [61]. It has an estimated incidence rate of lies occurred in 58%, of whom 2, 3 and 4 additional 1:60,000, and a male:female ratio of 2:7. Almost all malformations were observed in 50%, 29%, and 16% cases are sporadic with recurrence risk similar to of cases, respectively. Only three patients showed the that of the general population [49]. It was originally full picture of VACTERL. In another study of 140 pa- believed that sirenomelia is a severe form of CRS; tients with ARM, only 2 patients exhibited all of the however it has since been suggested that it is rather characteristics of the association, but 44 patients had the result of early embryonic vascular insult leading 3 or more of the components of VACTERL associa- to ischaemia in the caudal portion of the foetus [5, tion besides the ARM [76]. 15, 117]. It is thought that in sirenomelia an aberrant This association is frequent and it is estimated to vessel originating from the vitelline artery shunts the be 1:7,000–10,000 live births [21]. Almost all cases blood supply coming to the high abdominal aorta di- are sporadic and the recurrence risk is minimal. rectly through the umbilical cord to the placenta. The However, VATER with hydrocephalus represents a result would be severe hypoperfusion of structures distinct entity, since an autosomal recessive mode of distal to the origin of that aberrant vessel, since this inheritance has been reported in several families [47, vessel steals the blood supply from the caudal region 119]. A novel germline mutation of the PTEN gene of the foetus; this vascular stealing phenomenon is in a patient with macrocephaly, ventricular dilata- thought to be responsible for the pathogenesis of si- tion and features of VATER association was recently renomelia [112]. reported [94]. Another form of VACTERL and hydrocephalus is thought to be X-linked recessive [32, 46, 65]. Chromosomal anomalies have been reported frequently in the literature in association with VAC- 2 Genetics of Anorectal Malformations 23

2.4.4.6 Caudal Regression Syndrome mary or secondary neurulation, or differentiation are compromised in the embryonic caudal parts [15, 113, As the name implies, this syndrome is characterised 118, 127]. by a heterogeneous group of caudal anomalies. It may include variable degrees of spinal column agenesis, ARM and genitourinary anomalies. Its effects are not 2.4.4.7 Currarino Syndrome restricted to the caudal part of the body; CRS can also be associated with pulmonary hypoplasia and CS was first described as the triad of ARM, hemisa- congenital heart malformations [29]. The estimated crum and presacral mass [23]. The radiological aspect incidence rate of CRS is 1:7,500 births, but some of the sacrum described by Currarino is the so-called authors report a higher incidence of 1:200–1:1,000 “sickle-shaped sacrum”, which is caused by the pres- [125], while Diel et al. [26] reports an incidence rate ence of a hemisacrum with preservation of the first of 1:10,000–1:20,000. We believe that the difference in sacral vertebra. This finding is pathognomonic for previously reported incidence rates is due to the vari- the diagnosis CS [103]. According to Cama [16], ous presentations of CRS and to the possibility of its classification of sacral anomalies presents: (1) total association with other multisystemic malformations sacral agenesis with normal or short transverse pelvic such as omphalocele, cloacal exstrophy, imperforate diameter, and the defect extending to include some anus, and spinal deformities, and VACTERL [104]. lumbar vertebrae, (2) total sacral agenesis with intact Cama et al. [16] outlined some of the typical fea- lumbar vertebrae, (3) partial agenesis or hypodevel- tures of this syndrome: (1) cutaneous signs (such oped sacrum (preserved S1), (4) hemisacrum and flattening of the buttock and shortening of the inter- (5) coccygeal agenesis. Following this classification, gluteal cleft secondary to lumbosacral agenesis), (2) CS has been characterised as a type 4 sacral anomaly. sacrococcygeal agenesis (partial or total), (3) skel- Different forms of ARM can be present in CS, such etal deformities (vertebral, rib, or even lower-limb as rectourethral fistula, rectovestibular fistula and deformities, scoliosis with or without kyphosis, hip rectocloacal fistula [73]. It is estimated that 29% of dislocation), (4) congenital heart defects (tetralogy of ARM are associated with sacral anomalies [97]. The Fallot), (5) ARM, (6) genitourinary disorders (renal most frequent ARM in CS is ARM with perineal fis- aplasia or dysplasia, whether unilateral or bilateral, tula [86]. The same anomaly was reported by some vesicoureteral reflux, ureterocele, hypospadias and investigators as anorectal stenosis [84]. The presence malformed external genitalia) and (7) pulmonary hy- of perineal fistula leads to difficulty in defecation, and poplasia. ARM is considered a frequent finding with constipation, which can be the leading symptom in CRS: 27–48% of published CRS series of cases were the diagnosis of the disease [73]. The presacral mass associated with ARM [16, 96]. ARM can be present in can be an anterior meningocoele, teratoma, dermoid CRS in different forms, whether mild or severe. cyst, rectal duplication, a combination of these or an- Nearly all the cases of CRS are sporadic, and the other uncommon tumour with reported malignant genetic background to the development of CRS is forms [81, 114]. partially known [15, 113, 127]. CRS occurs in up to This disorder can be associated with other defects, 1% of pregnancies of diabetic women, and up to 22% such as urologic abnormalities including horseshoe, of CRS occurs in the offspring of mothers affected duplex, or dysplastic kidney, vesicoureteric reflux, with diabetes mellitus type I or type II [118]. The risk duplex ureter or hypospadias. Gynaecological mal- seems to be greater for women who are insulin-de- formations may include bicornuate uterus, septate pendent since it is estimated that they are 200–400 vagina and bifid clitoris. Thus, the term syndrome times more likely to have a child with CRS than non- was applied correctly rather than triad [4, 67]. CS can diabetic women, reflecting the fact that CRS is one be sporadic or familial, with an autosomal dominant of the most characteristic abnormalities occurring in mode of inheritance. It has variable expression (i.e. it foetuses of diabetic women. A reasonable explana- can present with all or some of the previously men- tion of this combination is that the teratogenic cause tioned anomalies), and incomplete penetrance, which underlying CRS in diabetes is hyperglycaemia [33]. means that some individuals have the affected geno- To the best of our knowledge, the exact mechanism type but with normal phenotype. In the biggest series leading to CRS is not yet known; however, it has been in the literature, the female:male ratio is 1.7:1, with proposed that before the 7th week of gestation, one the possibility that the greater number of females is or more processes of primitive streak migration, pri- due to the coexistence of the gynaecologic or urologic 24 Giuseppe Martucciello

problems, which are noticed more frequently in fe- mutations causing CS are in total 9 missense, 2 non- males. Moreover, 33% of cases maybe asymptomatic sense, 2 splicing, 7 frameshifts, and 6 hemizygous [67]. microdeletions. Since the loss of one copy of HLXB9 Previous studies suggest the importance of a locus gene (haploinsufficiency) can lead to CS, as well as on chromosome 7q39. This region was identified by cases involving deletions of the region 7q35-tel, it is linkage analysis to search for the causative gene for quite probable that CS is caused by loss-of-function CS [3, 66]. The region 7q39 includes three genes; one mutations. The presence of polyalanine expansion of the best known is Sonic Hedgehog (SHH), a very with various triplet repeats in homeobox genes can important molecular factor in early embryogenesis lead to certain pathologic conditions, for example in different body systems; its mutations are respon- synpolydactyly, oculopharyngeal muscular dystrophy, sible for holoprosencephaly in humans [3]. SHH was and cleidocranial dysplasia [13, 34, 79]. This abnor- excluded as a causative gene for CS by two thorough mal expansion was not found to determine CS, since investigations performed by Seri et al. [107] and Var- they were found in both affected and control cases. gas et al. [120]. The remaining two homeobox genes In the study performed by Belloni et al. [4], the com- are also important for early development, namely monest allele in the heterozygous form for the general EN2 and HLXB9. Further linkage analysis on affected population was CGC11, accounting for 90.23% of the families with sacral agenesis showed that the position 100 total control chromosomes. Other cases observed of the causative gene was more towards the terminal were CGC12, CGC9 and CGC8, accounting for 1.7, end of the long arm of chromosome 7. This excluded 7.47 and 0.6%, respectively. Only one case was found more obviously the involvement of SHH and EN2, with homozygous change in the polyalanine tract giv- which are located more centromerically. EN2 was ex- ing rise to the CGC9/CGC9 allelic combination. The cluded since it is located upstream of SHH [101, 107]. sample was a “control” case, but a closer look and de- The third candidate gene is HLXB9 (homeobox gene), tailed investigations showed that the person lacked which was found to be expressed in early development posterior arch fusion of the vertebrae and had left- in human embryos and expressed specifically in the sided scoliosis. The possible relationship between anterior horn region of the spinal cord [101]. HLXB9 the length of the alanine fragment and the presence is composed of three exons. A combination of DGGE, of sacral anomalies in either affected or control cases single-strand conformation polymorphism and di- was also excluded in that study [4]. rect DNA sequencing experiments were carried out. The same studies showed in some familial cases, These studies demonstrated several mutations in the that the mutation was found as well in asymptomatic gene coding sequence and in intron-exon boundaries patients with normal sacral X-ray giving the disease in both sporadic and familial cases, confirming the the characteristic of incomplete penetrance. HLXB9 causative role of HLXB9 in this syndrome (Fig. 2.2) mutations are not found in other forms of sacral [4, 36, 101]. To the best of our knowledge, the known agenesis and are found only in individuals with CS. However, HLXB9 mutations are not found in all patients who are diagnosed clinically; this may suggest the possibility of genetic heterogeneity or the presence of some non-genetic components influencing the occurrence of this anomaly, at least for sporadic cases. To the best of our knowledge, no further studies have proved the involvement of any other genes in sacral and anorectal development [104]. However Horn et al. [45] have reported four cases of minimal clinical expression of holoprosencephaly and CS due to different cytogenetic rearrangements affecting both SHH and HLXB9 at 7q36.3 Taking into consideration all of the previous data, Fig. 2.2 Schematic representation of the structure of HLXB9 we believe that genetic counselling is highly appreci- gene, showing the three exons, the homeodomain, the number- ated in any diagnosed case of CS. There is always the ing according to genomic sequence, and nucleotide numera- need for detailed physical examination and sacral X- tion according to cDNA sequence, and residues numeration. ray to the parents to exclude minimal signs of the dis- The arrows indicate the sites of discovered mutations along the ease. Finding HLXB9 mutations in the index case ne- gene cessitates the molecular genetic study of the parents, 2 Genetics of Anorectal Malformations 25

Table 2.4 Examples of genetic syndromes with ARM as a feature. XR X-linked recessive, AD autosomal-dominant, AR autosomal- recessive Syndrome Prominent Features Mode of Locus or gene inheritance if known Opitz G Hypertelorism, hypospadias, swallowing difficulties. XR, MID 1 gene Opitz Frias The same features of Opitz AD 22q11.2 Fraser cryptophthalmos with other malformations, AR FRAS1 cryptophthalmos-syndactyly syndrome. Johanson-Blizzard Hypoplastic alae nasi, deafness, pancreatic AR - insufficiency, hypothyroidism. CHARGE Coloboma, heart anomaly, choanal atresia, Sporadic or AD CHD7 retardation, genital and ear anomalies EEC Ectrodactyly, ectodermal dysplasia, and cleft lip/palate AD P63 Goldenhaar Hemifacial microsomia, cardiac, vertebral, Sporadic or AD and central nervous system defects Velocardiofacial Cardiac, thymic, hypocalcaemia, vertebral, others Sporadic or AD Chromosome 22 microdeletion McKusick-Kaufman Hydrometrocolpus, Hirschsprung, hydronephrosis AR MKKS

even if they were asymptomatic, because of incom- induced rats showed the presence of maldevelopment plete penetrance with possible subsequent extension of the cloaca, delayed tailgut regression, increased of the study for further family members in the case posterior cloacal wall apoptosis, and underdevelop- of positive results. Since CS is autosomal dominant ment of the dorsal aspect of the cloaca itself and its and the recurrence risk is 50%, the disease has both membrane [90]. incomplete penetrance and variable expression. Pre- Although SHH mutations were excluded as a cause natal diagnosis through ultrasound can only detect of ARM in humans [107, 120], Shh and the Shh-re- the presacral mass, which is not present in all cases, sponsive transcription factors Gli2 and Gli3 have making the molecular diagnosis the most favourable proved to be essential for mammalian foregut de- method for diagnosing CS prenatally [67]. ARM can velopment. Mutant mice for these factors showed a also be associated with many other syndromes, al- series of multisystemic defects including ARM and though in some cases it maybe not be a main charac- other components of VACTERL association in man teristic feature (Table 2.4) (see Chap. 3). [54, 63, 77, 78, 92]. The ablation of Hlxb9 expression was studied in two murine knockout models. Heterozygous mice had 2.5 Mouse Models with ARM no apparent abnormalities, but the homozygous mu- tants Hlxb9 -/- were incompatible with life, although ARM can be induced by using certain teratogens such they exhibited no obvious morphologic abnormality. as ethylenethiourea (ETU) [41], retinoic acid [28], Differential models were made for the cartilage and and adriamycin [64] in mice, rats [59] and piglets bones, with normal skeletal development and absent [90]. The ARM produced by adriamycin in rat foe- sacral defects. However, defects were described in the tuses seem to be more severe and include variations development of the pancreas, which is not seen in hu- of cloacal agenesis, which is not generally considered mans affected with CS [38, 62] (Table 2.5). the best example with which to study comparable human ARM [27]. ETU treatment of timed-pregnant rats showed ARM in 80% of offspring, with variable 2.6 Conclusions phenotypes ranging from simple anal membrane to rectourethral fistula, resembling the ARM seen in the Current knowledge of the clinical genetics, cytogenet- human spectrum [41]. Recently, an explanation of ics and molecular genetics of ARM is still progressing. the developmental defects leading to ARM in ETU- The genetic basis of these anomalies is very complex 26 Giuseppe Martucciello

Table 2.5 Human and mouse genotypic/phenotypic correlations. GCPS Greig cephalopolysyndactyly syndrome, PHS Pallister- Hall syndrome Gene mutation Effects in man Effects in animal model HLXB9 Currarino syndrome Hlxb9: Heterozygous mice (normal skeleton except (Heterozygous mutation) for curled body and small size) Homozygous mutation (incompatible with life, defects in motor neurons). SALL1 Townes-Brocks syndrome Sall1: Heterozygous mice (no detectable phenotype) (heterozygous mutation) Homozygous mutation (all died in neonatal period, only renal anomalies) GLI3 PHS and GCPS (het- Gli3 mutant mice shows similar phenotype to PHS and GCPS erozygous mutation) SHH Holoproscencephaly Shh and/or its transcription factor mutations: different malformations (heterozygous mutations) including gastrointestinal tract malformations (ARM)

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