Clinical aspects of defects in the determination of laterality

PCD/Kartagener

One fourth to one fifth of individuals with complete, mirror-image situs inversus have Kartagener syndrome. The classic Kartagener syndrome triad of clinical features includes situs inversus, bronchiectasis, and chronic sinusitis. Primary ciliary dyskinesia (PCD) or immotile cilia syndrome describes a causally heterogeneous group of autosomal recessive disorders characterized by partial or complete deficiency of ciliary motility associated with a variety of ultrastructural abnormalities such as absent dynein arms, absent radial spokes, and disturbed ciliary orientation [Rott, 1979; Afzelius, 1981; Tonkin, 1984; Afzelius and Mossberg, 1995; Tkebuchava et al., 1996; Jorissen et al., 1997; Marszalek et al., 1997; Teknos et al., 1997]. The characteristic phenotype includes bronchiectasis with chronic cough, rhinitis, sinusitis, male infertility caused by sperm immotility, and variable impairment of female fertility. Complete, mirror-image situs inversus is present in only one half of these patients [Moreno and Murphy, 1981]. Even a pair of monozygotic twins with PCD has been found to be discordant for situs inversus, suggesting that it is truly a random process [Noone et al., 1999].

Because approximately 20–25% of individuals with situs inversus have symptoms suggestive of ciliary dysfunction, the incidence of Kartagener syndrome appears to be approximately 1/120,000 to 1/40,000 [Afzelius and Mossberg, 1995]. Therefore, the overall prevalence of immotile cilia syndromes or PCD may be in the range of 1/60,000 to as high as 1/20,000, because only half of these individuals have situs inversus. PCD occurs in all human races [Rott, 1979].

Recent mapping studies suggest extensive locus heterogeneity in the European and North American population, with potentially interesting regions on 3p, 4q, 5p, 7p, 8q, 10p, 11q, 13q, 15q, 16p, 17q, and 19q [Blouin et al., 2000]. Most suggestive evidence for linkage occurred on 8q, 16pter, 19q. In an Arab population, significant linkage has been found for a locus on 19q [Meeks et al., 2000].

A human PCD gene has been identified using a candidate-gene approach. A human sequence (DNAI1) related to a Chlamydomonas gene necessary for outer dynein arms was isolated, and one patient was identified with two loss-of-function mutations [Pennarun et al., 1999]. That patient had PCD with immotile respiratory cilia that lacked outer dynein arms. Subsequently, two more families have been found with PCD and DNAI1 mutations [Zariwala et al., 2000].

Because a large number of axonemal dynein proteins are involved in the structure and function of cilia, they are obvious candidate genes of great research interest for these causally heterogeneous PCD disorders. For example, in a consanguineous Lebanese family with ciliary akinesia, situs inversus, and absent outer dynein arms by electron microscopy, homozygosity mapping has identified significant linkage on chr 5p15-p14 [Omran et al., 2000]. An excellent candidate gene in that region of linkage is DNAH5, which has high homology to the axonemal Chlamydomonas gamma-heavy dynein chain.

Malformations Associated With Polyasplenia

Patients with asplenia or polysplenia usually have other, severe malformations that are life threatening. All thoracic and abdominal organ systems may be affected. The list of associated malformations is extensive and involves virtually every thoracic and abdominal organ system [Aylsworth, 1993; Splitt et al., 1996]. As many as 90% of patients with polysplenia and 99% of patients with asplenia are reported to have congenital heart disease, but because many patients are ascertained because of major malformations such as cardiac anomalies, prevalence data for these associated anomalies may be biased. In addition to the usual spectrum of thoracic and visceral malformations, there is also good evidence that central nervous system anomalies and defects of the axial skeleton are nonrandomly associated with the polyasplenia spectrum.

Abdominal heterotaxy in the asplenia phenotype includes bowel malrotation, a symmetric liver, and right-sided stomach. Extracardiac malformations are common and frequently involve the gastrointestinal, genitourinary, bronchopulmonary, axial skeletal, and central nervous systems [Putschar and Manion, 1956; Freedom, 1972]. Cardiovascular malformations are usually conotruncal and tend to be more severe than those associated with the polysplenia phenotype [Polhemus and Schafer, 1952; Ivemark, 1955; Putschar and Manion, 1956; Ruttenberg et al., 1964; Campbell and Deuchar, 1967; Van Meirop et al., 1972; Freedom, 1974; Rose et al., 1975; Majeski and Upshur, 1978; Tonkin, 1984; Winer-Muram and Tonkin, 1989]. The inferior vena cava and the abdominal aorta usually have an anomalous relationship, both lying on the same side of the spine, regardless of the side occupied by the vena cava. This relationship was thought to be pathognomonic for asplenia until it was observed in a patient with polysplenia [Freedom, 1974].

Patients with the polysplenia phenotype also have bowel malrotation and a liver that is symmetrical or inverted with the larger lobe on the left. The stomach is usually right sided. Two or more spleens are frequently present along the greater curvature of the stomach. Cardiovascular malformations most commonly include malposition, atrial septal defects, ventricular septal defects, bilateral superior vena cavae, partial anomalous pulmonary venous return, and intrahepatic interruption of the inferior vena cava with connection to the azygous or hemiazygous vein [Moller et al., 1967; Van Meirop et al., 1972; Rose et al., 1975; Hallett et al., 1979; Peoples et al., 1983; Winer-Muram and Tonkin, 1989]. Extracardiac malformations are common as in asplenia and also involve the gastrointestinal, genitourinary, bronchopulmonary, axial skeletal, and central nervous systems.

Extrahepatic biliary atresia (EHBA) is uniquely associated with polysplenia, and it has been estimated that 10–20% of patients with EHBA have associated malformations like those seen in polysplenia [Chandra, 1974; Maksem, 1980; Falchetti et al., 1991; Silveira et al., 1991; Davenport et al., 1993; Vazquez et al., 1995; Kataria et al., 1996; Nakada et al., 1997; Tanano et al., 1999]. Interestingly, the inv mouse model for situs inversus has extrahepatic biliary obstruction, similar to that seen associated with polysplenia [Mazziotti et al., 1999].

Asplenia rarely is reported to occur without other severe malformations in individuals with situs solitus or complete situs inversus [Putschar and Manion, 1956; Muir, 1969; Waldman et al., 1977; Hauser and Silberman, 1982]. Some cases are familial involving sibs [Gillis et al., 1992] and parent–child pairs [Lindor et al., 1995]. One of the father–son pairs described by Lindor and colleagues included a mild form of cardiac atrial isomerism in one child, suggesting that isolated asplenia may represent the mild end of the polyasplenia spectrum.

Asymptomatic polysplenia with heterotaxia but without severe or complex cardiac or visceral malformation also occurs [Peoples et al., 1983; Gayer et al., 1999]. Note that one or more small, accessory spleens may occasionally be found in individuals with situs solitus. They are usually asymptomatic and not considered to be caused by defects in the determination of laterality, but may be associated with other malformations such as splenogonadal fusion and limb reduction.

Mendelian Factors

Observations of families with affected sibs led to the suggestion of autosomal recessive causation [Polhemus and Schafer, 1952; Chen and Monteleone, 1977; Zlotogora and Elian, 1981; Toriello et al., 1986]. Even better evidence for autosomal recessive inheritance comes from reports of affected sibs who have consanguineous parents [Arnold et al., 1983; Gatrad et al., 1984; Czeizel, 1987]. The recessive nature of situs inversus associated with ciliary dyskinesia is discussed elsewhere.

Situs ambiguus and complete, mirror-image situs inversus (thoracic and/or abdominal, with or without ciliary abnormality) may be parts of the same spectrum of malformation-association. One boy and two of his cousins had typical Kartagener syndrome with abnormal cilia and situs inversus. His sister, who also displayed respiratory symptoms associated with abnormal cilia, had features of situs ambiguus including pulmonic stenosis, absence of the inferior vena cava with azygous continuation, midline liver, and two contiguous splenic masses, suggesting that at least one of her predisposing factors was a ciliary dyskinesia gene [Schidlow et al., 1982].

An interesting family suggesting autosomal dominant inheritance, or at least a vertically transmitted susceptibility gene, contained four individuals from three generations with laterality defects. Two individuals had complete situs inversus, and two had situs ambiguus (asplenia, midline liver, and complex cardiac malformation) [Casey et al., 1996]. Two obligate carriers were anatomically normal. Several other reports also suggest the existence of either straightforward autosomal dominant inheritance of situs inversus and situs ambiguus, or at least a monogenic predisposition to laterality defects running through families [Niikawa et al., 1983; Gershoni-Baruch et al., 1989; Alonso et al., 1995].

As mentioned above, individuals with mirror-image situs inversus usually have a low incidence of structural malformations, but occasional families have a high frequency of other anomalies, in a manner reminiscent of the iv mouse (v.i.) [Zlotogora et al., 1987]. Some families have had recurrence of the malformations that are typically associated with situs ambiguus, such as congenital heart disease and polyasplenia, with variable occurrence of complete situs inversus or other features of situs ambiguus [Silver et al., 1972; Katcher, 1980; Arnold et al., 1983; Distefano et al., 1987; Zlotogora et al., 1987; Mikkila et al., 1994; Alonso et al., 1995].

These observations are of particular interest because they suggest that isolated birth defects such as congenital heart disease, anal atresia, diaphragmatic hernia, bicornuate uterus, etc. may be caused by predisposing genes that are involved in left–right axis determination pathways. In fact, one such gene has been identified. The zebrafish oep gene is involved in left–right axis determination [Yost, 1998a, b] and the mouse homolog, Cryptic, is also important for laterality determination [Gaio et al., 1999; Yan et al., 1999]. Recently, three missense and one frameshift mutation have been found in the human CRYPTIC gene in four patients with situs inversus and five with situs ambiguus [Bamford et al., 2000b]. Subsequent studies have identified two of these mutations, along with one other new mutation, in patients who have only transposition of the great arteries, i.e., isolated congenital heart disease without any other sign of abnormal situs determination [Bamford et al., 2000a]. Therefore, as predicted, at least some patients with isolated, nonsyndromic conotruncal heart defects have their malformations caused by mutations in a gene involved in left–right axis determination. They do not happen to have diagnostic features of situs ambiguus such as polyasplenia or heterotaxy, perhaps by chance or because of the effects of other genes or environmental factors. We predict that other isolated, nonsyndromic malformations will turn out to be caused by similar mechanisms of mutation involving genes in pathways of situs determination.

In 1987, Mathias et al. reported a family in which nine males in two generations were affected by a spectrum of abnormalities of laterality, including congenital heart disease, situs inversus, or polyasplenia and an X-linked recessive inheritance pattern. All 11 affected relatives had some form of situs inversus or situs ambiguus. Both asplenia and polysplenia occurred, with cardiac, central nervous system, and vertebral malformations, including sacral dysplasia with anorectal anomalies [Mathias et al., 1987].

Another X-linked family has the unique finding of situs inversus in carrier females and situs ambiguus in males [Gebbia et al., 1997]. The affected males had lethal malformation syndromes associated with situs ambiguus, whereas carrier females had mirror-image situs inversus or situs solitus. Anal and sacral anomalies occur in both affected males and carrier females.

The X-linked family described by Mikkila et al. [1994] also features caudal anomalies in the affected males and genitourinary anomalies in carrier females (bicornuate uterus, uterine septum, septate vagina and uterus), and one had anal stenosis.

Several other cases of situs ambiguus with caudal dysgenesis have been described [Peoples et al., 1983; Fullana et al., 1986; Rodriguez et al., 1991]. We have studied a pair of brothers with situs ambiguus and complex congenital heart disease, one of whom had sacral dysplasia and imperforate anus. It seems likely that most isolated cases of situs inversus or situs ambiguus with caudal dysplasia and/or anal anomalies may be caused by X-linked causes of laterality defects, because familial recurrences of this association are reported only in families compatible with X-linked inheritance.

The causes of this phenotype of X-linked situs ambiguus, with variable manifestations of caudal dysplasia in both hemizygous males and heterozygous females, and variable expression of complete, mirror-image situs inversus in heterozygous females, have been shown to be various mutations in the X-linked gene, ZIC3 [Ferrero et al., 1997; Gebbia et al., 1997]. Zic3 is also the gene deleted in the X-linked mouse mutation Bent Tail, which causes kinked tails because of aberrant tail bone development, exencephaly in some, and situs ambiguus in some of both the hemizygous males and heterozygous females [Carrel et al., 2000; Klootwijk et al., 2000] The Zic3 knockout mouse has a phenotype similar to the human ZIC3 mutations, including situs ambiguus, central nervous system malformations (exencephaly), and axial skeletal anomalies (vertebral dysplasia, fusion, and duplication) [Purandare et al., 2000; Casey, 2001].

Chromosome Abnormalities

Patients reported with situs inversus or situs ambiguus and various chromosome abnormalities are listed in Table I. Chromosome rearrangements can cause overexpression of duplicated genes, deficiency of deleted gene products, or disruption of single genes. Therefore, patients with laterality defects and chromosomal abnormalities may be useful in mapping genes involved in left–right axis determination. Conversely, it should be kept in mind that associations seen in only a single patient may be coincidental.

Teratogens

Renal–Hepatic–Pancreatic Dysplasia and Skeletal Dysplasia

Ivemark, who described the syndrome of asplenia, also described a familial condition characterized by renal–hepatic–pancreatic dysplasia (RHPD) [Ivemark et al., 1959]. This Ivemark RHPD phenotype was subsequently observed to occur in association with situs ambiguus, and there is evidence that at least some RHPD and RHPD with situs ambiguus is autosomal recessive [Crawfurd, 1978; Lurie et al., 1991; Torra et al., 1996].

A lethal syndrome of cystic renal dysplasia, pancreatic cysts with interstitial fibrosis, intrahepatic biliary dysgenesis, and total situs inversus was described in two unrelated patients who were born 8 years apart in the same remote Japanese town [Yoshikawa et al., 1981; Hiraoka et al., 1988]. Another three sibs with situs inversus totalis and severe cystic dysplasia of the kidneys and pancreas have been subsequently described, but they had no liver disease. They also had bowing of the lower limbs and clavicles, and intrauterine growth retardation [Balci et al., 1999, 2000]. These patients seem to have a syndrome similar to the RHPD described above, but with complete situs inversus instead of polyasplenia. Other reports of patients with RHPD associated with situs inversus include one of the patients described by Bernstein in his review of RHPD [Bernstein et al., 1987].

Spranger described a patient with situs inversus and renal and pancreatic dysplasia associated with the Saldino-Noonan type of short rib–polydactyly syndrome [Spranger et al., 1974]. A patient, reported by Fraser, had situs inversus, typical radiographic features of chondroectodermal dysplasia, and a number of other anomalies including cleft epiglottis and larynx, skin tags on the neck, postaxial hexadactyly of hands and feet, pancreatic cystic dysplasia, renal dysplasia, ambiguous genitalia with micropenis, and imperforate anus [Fraser et al., 1989]. A patient described by Brueton and colleagues had skeletal features of Jeune asphyxiating thoracic dystrophy with situs inversus, and RHPD [Brueton et al., 1990]. Brueton and colleagues also pointed out that older patients with Jeune syndrome commonly have some form of RHPD. In that same article, Brueton et al. described another patient with skeletal features of chondroectodermal dysplasia along with situs inversus but no RHPD, very much like a patient reported by Donlan et al. [1969]. Several other patients have been reported with short rib-polydactyly syndromes and situs inversus or situs ambiguus [Belloni and Beluffi, 1981; Bernstein et al., 1985; de Sierra et al., 1992; Tsai et al., 1992; Urioste et al., 1994; Digilio et al., 1999]. At least one patient with Jeune syndrome and situs abnormalities has been observed [Majewski et al., 1996]. The association of Jeune syndrome with RHPD-like complications is well known in those patients who survive through later childhood.

An analysis of the congenital heart defects seen in “oral–facial–skeletal syndromes” (short rib–polydactyly syndromes and other possibly related oral–facial–skeletal syndromes) showed a pathogenetic similarity to the types of heart defects seen in the polyasplenia spectrum [Digilio et al., 1999]. These overlapping phenotypes suggest an interesting community of syndromes (Fig. 1). Although most patients with SRP or RHPD do not have associated laterality defects, these phenotype associations appear to be noncoincidental, and may provide clues about underlying causes (i.e., genes and/or environmental factors), or common pathogenetic sequences (i.e., interactions between developmental pathways).

Situs Ambiguus With Campomelia, RHPD, and Cervical Lymphocele

Campomelia, cervical lymphocele, polycystic kidneys, pancreas, and liver, short gut, and polysplenia have been described in sibs [Cumming et al., 1986] and in another unrelated patient [Ming et al., 1997]. Skeletal, lymphatic, and renal lesions were seen at 26 weeks' gestation by ultrasonography, but not at 16 weeks. Sibs with campomelia, polycystic dysplasia, and cervical lymphocele but without polysplenia have also been described [Urioste et al., 1991].

Agnathia–Holoprosencephaly

A lethal malformation complex of agnathia with associated anomalies has been reported in a number of patients, 10 of whom have had situs inversus and/or situs ambiguus [Pauli et al., 1981; Leech et al., 1988; Hersh et al., 1989; Robinson and Lenke, 1989; Meinecke et al., 1990; Persutte et al., 1990; Stoler and Holmes, 1992; Ozden et al., 2000]. Other malformations reported to be associated have involved the genitourinary, gastrointestinal, and central nervous system. Five of the patients with agnathia and situs abnormality have had holoprosencephaly. Three of these had situs inversus totalis and two had situs ambiguus with polysplenia. Other patients with a similar but less severe pattern of malformation are those with micrognathia and aglossia/hypoglossia, and several have had situs inversus [Hussels, 1971; Dunham and Austin, 1990]. Finally, two sibs, a brother and sister of consanguineous parents, were reported to have situs ambiguus with associated meningomyelocele in the female and holoprosencephaly in the male [Bonneau et al., 1999]. These overlapping phenotypes suggest another interesting potential community of syndromes (Fig. 2). Although most patients with agnathia or holoprosencephaly do not have associated laterality defects, these associations appear to be noncoincidental, and, like the SRP-RHPD group, their analysis should provide clues about common underlying causes or pathogenetic sequences.

The iv Mouse Model

Approximately half of mice homozygous for the iv (situs inversus) mutation have situs inversus [Hummel and Chapman, 1959; Layton, 1976]. Although this mouse model is frequently thought of as a model for complete, mirror-image situs inversus, it should be noted that as many as 40% of the homozygotes also have some type of heterotaxy or visceral malformation. Homozygous iv mice have a variable but frequent occurrence of malformation, most notably of the cardiovascular system but also involving lung lobation, liver location, bowel rotation, and spleen shape, size, presence, and location. Some early embryos have markedly distorted bulboventricular loops. Layton proposed that the heterotaxia and associated malformations can all be explained by defective control of the shape of the cytoskeleton and cytomusculature, which appears to be important in the early formation of the cardiac loop [Layton, 1978]. The iv mouse model seems more like some of the rare families reported that include both situs inversus and situs ambiguus [Zlotogora et al., 1987] than it does like pure situs inversus without heterotaxy or malformation.

The iv-/- phenotype is not associated with respiratory ciliary abnormalities [Handel and Kennedy, 1984]. It is, however, caused by a mutation in a gene coding for an axonemal dynein heavy chain, left/right-dynein (lrd) that appears to be expressed in the monocilia of the node during gastrulation, but not later in respiratory cilia [Supp et al., 1997, 1999; Wagner and Yost, 2000]. The nodal cilia, in turn, appear to be important in initiating normal left–right axis asymmetry determination [Marszalek et al., 1999; Supp et al., 2000].

The mouse gene that codes for lrd, Dnahc11, was originally mapped to mouse chromosome 12 [Hanzlik et al., 1990]. The human gene maps to chromosome 7 [Chapelin et al., 1997; Bartoloni et al., 1999]. Recently, a homozygous R to X nonsense mutation in an exon coding for the motor domain of the human gene, DNAH11, has been identified in a patient with situs inversus [Bartoloni et al., 2000]. The patient also had cystic fibrosis caused by paternal uniparental disomy for chromosome 7, abnormal ciliary motility by direct observation of wet preparations from a bronchial biopsy, but structurally normal cilia by electron microscopy (as is seen in the iv-/- mouse) [Pan et al., 1998]. Bartoloni et al. commented that they do not expect a significant fraction of patients with PCD to have mutations in this gene because their linkage analysis in 50 nuclear families did not show a significant lod score on chromosome 7p.