Characterization of phenotypic spectrum of fetal heterotaxy syndrome by combining ultrasound and magnetic resonance imaging.

OBJECTIVE: Heterotaxy or isomerism of the atrial appendages is a congenital disorder with variable presentation, associated with both cardiac and non-cardiac anomalies, which may have a serious impact on fetal outcome. The aim of this exploratory study was to assess the value of fetal magnetic resonance imaging (MRI), as a complementary tool to ultrasound, for describing the morphological spectrum encountered in heterotaxy. METHODS: This retrospective study included 27 fetuses that underwent fetal MRI following prenatal suspicion of heterotaxy on ultrasound from 1998 to 2019 in a tertiary referral center. Heterotaxy was classified as left atrial isomerism (LAI) or right atrial isomerism (RAI) based on fetal echocardiography (FE) examination. In addition to routine prenatal ultrasound, fetal MRI was offered routinely to enhance the diagnosis of non-cardiac anomalies, which might have been missed on ultrasound. Prenatal findings on ultrasound, FE and MRI were reviewed systematically and compared with those of postnatal imaging and autopsy reports. RESULTS: Twenty-seven fetuses with heterotaxy and cardiovascular pathology, of which 19 (70%) had LAI and eight (30%) had RAI, were included. Seven (7/19 (37%)) fetuses with LAI had normal intracardiac anatomy, whereas all fetuses with RAI had a cardiac malformation. All 27 fetuses had non-cardiac anomalies on fetal MRI, including situs and splenic anomalies. In 12/19 (63%) fetuses with LAI, a specific abnormal configuration of the liver was observed on MRI. In three fetuses, fetal MRI revealed signs of total anomalous pulmonary venous connection obstruction. An abnormal bronchial tree pattern was suspected on prenatal MRI in 6/19 (32%) fetuses with LAI and 3/8 (38%) fetuses with RAI. CONCLUSIONS: Visualization on MRI of non-cardiac anomalies in fetuses with suspected heterotaxy is feasible and can assist the complex diagnosis of this condition, despite its limitations. This modality potentially enables differentiation of less severe cases from more complex ones, which may have a poorer prognosis. Fetal MRI can assist in prenatal counseling and planning postnatal management. © 2021 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Loss of ciliary transition zone protein TMEM107 leads to heterotaxy in mice.

Cilia in most vertebrate left-right organizers are involved in the original break in left-right (L-R) symmetry, however, less is known about their roles in subsequent steps of the cascade - relaying the signaling and maintaining the established asymmetry. Here we describe the L-R patterning cascades in two mutants of a ciliary transition zone protein TMEM107, revealing that near-complete loss of cilia in Tmem107null leads to left pulmonary isomerism due to the failure of the midline barrier. Contrary, partially retained cilia in the node and the midline of a hypomorphic Tmem107schlei mutant appear sufficient for the formation of the midline barrier and establishment and maintenance of the L-R asymmetry. Despite misregulation of Shh signaling in both mutants, the presence of normal Lefty1 expression and midline barrier formation in Tmem107schlei mutants, suggests a requirement for cilia, but not necessarily Shh signaling for Lefty1 expression and midline barrier formation.

Fetal case of right atrial isomerism with infracardiac total anomalous pulmonary venous connection and agenesis of the ductus venosus.

After birth, the ductus venosus becomes an important route connecting the pulmonary and systemic venous systems for survival in infracardiac total anomalous pulmonary venous connection. We encountered a fetal case of right atrial isomerism with infracardiac total anomalous pulmonary venous connection and agenesis of ductus venosus. Prenatal echocardiography suggested that the fetus had severe pulmonary venous obstruction; however, no obstructive lesions were detected at the level of the vertical vein that drained into the portal veins. Therefore, we concluded that emergency surgical pulmonary venous obstruction release was the only way for the fetus to survive. However, the saturation level was maintained above 70% due to the abundant communications via the hepatic sinusoid over 1 week after birth. In conclusion, hepatic sinusoids can be a sufficient route for pulmonary venous return and may not cause severe pulmonary venous obstruction in infracardiac total anomalous pulmonary venous connection with agenesis of ductus venosus.

ZIC3 in Heterotaxy.

Mutation of ZIC3 causes X-linked heterotaxy, a syndrome in which the laterality of internal organs is disrupted. Analysis of model organisms and gene expression during early development suggests ZIC3-related heterotaxy occurs due to defects at the earliest stage of left-right axis formation. Although there are data to support abnormalities of the node and cilia as underlying causes, it is unclear at the molecular level why loss of ZIC3 function causes such these defects. ZIC3 has putative roles in a number of developmental signalling pathways that have distinct roles in establishing the left-right axis. This complicates the understanding of the mechanistic basis of Zic3 in early development and left-right patterning. Here we summarise our current understanding of ZIC3 function and describe the potential role ZIC3 plays in important signalling pathways and their links to heterotaxy.

Comparing levocardia and dextrocardia in fetuses with heterotaxy syndrome: prenatal features, clinical significance and outcomes.

BACKGROUND: To investigate the differences in cardiovascular disease, extracardiac anomalies and outcomes between fetuses with levocardia and dextrocardia. METHODS: Clinical demographics, prenatal features, postnatal characteristics and the outcomes of fetuses with levocardia or dextrocardia were recorded and analyzed. RESULTS: Sixty-five fetuses with dextrocardia and thirty-eight fetuses with levocardia were enrolled. Right ventricle outlet obstruction, atrioventricular septal defect and intestinal malrotation were common in both groups. Univentricular physiology, transposition of the great arteries and esophageal atresia were more frequent in fetuses with levocardia, whereas abnormal pulmonary venous connection, double outlet of right ventricle, left ventricle outlet obstruction and brain abnormalities were more frequent in the dextrocardia group. The accuracy of evaluating cardiac malformations was high, but the sensitivity in assessing extracardiac abnormalities was low. CONCLUSIONS: Although the disorders have certain overlapping features, there are several differences between fetuses with levocardia and dextrocardia. These findings might improve patient counseling and perinatal management.

Extracardiac anomalies in prenatally diagnosed heterotaxy syndrome.

OBJECTIVE: To assess the incidence and impact of extracardiac anomalies on the prognosis of fetuses with heterotaxy syndrome. METHODS: All fetuses diagnosed with heterotaxy syndrome by three experienced examiners over a period of 14 years (1999-2013) were reviewed retrospectively. RESULTS: In total, 165 fetuses with heterotaxy syndrome were diagnosed in the study period. One hundred and fifty (90.9%) had cardiac defects; extracardiac anomalies that did not involve the spleen were present in 26/165 (15.8%) cases. Of the total study cohort, termination of pregnancy was performed in 49 (29.7%) cases, intrauterine death occurred in 11 (6.7%), postnatal death occurred in 38 (23.0%) and 67 (40.6%) were alive at the latest follow-up, resulting in a total perinatal and pediatric mortality of 59.4%. Among the 105 liveborn neonates, 15 (14.3%) had extracardiac anomalies with significant impact on the postnatal course: one neonate died following repair of an encephalocele, six had successful treatment for various types of intestinal malrotation and/or atresia and one underwent hiatal hernia repair; the remaining seven had biliary atresia, of which five died and the two survivors are awaiting liver transplantation. The status of the spleen was assessed in 93/105 liveborn children and was found to be abnormal in 84/93 (90.3%). There were three cases of lethal sepsis, all associated with asplenia. Of the 38 postnatal deaths, 29 (76.3%) had a cardiac cause, seven (18.4%) had an extracardiac cause and in two (5.2%) the reason was uncertain. CONCLUSIONS: Although the leading causes of death in fetuses and children with heterotaxy syndrome are cardiac, a small subset of fetuses have extracardiac anomalies with significant impact on outcome. These anomalies often escape prenatal detection, and therefore neonates at risk should be monitored for bowel obstruction, biliary atresia and immune dysfunction in order to allow timely intervention through a multidisciplinary approach. Copyright © 2015 ISUOG. Published by John Wiley & Sons Ltd.

Left-right asymmetry: cilia and calcium revisited.

Leftward flow generated by motile cilia is known to underlie left-right asymmetry in vertebrate embryos. A new study now links intraciliary calcium oscillations to cilia motility and the downstream nodal signaling cascade that drives left-sided development.

Intraciliary calcium oscillations initiate vertebrate left-right asymmetry.

BACKGROUND: Bilateral symmetry during vertebrate development is broken at the left-right organizer (LRO) by ciliary motility and the resultant directional flow of extracellular fluid. However, how ciliary motility is perceived and transduced into asymmetrical intracellular signaling at the LRO remains controversial. Previous work has indicated that sensory cilia and polycystin-2 (Pkd2), a cation channel, are required for sensing ciliary motility, yet their function and the molecular mechanism linking both to left-right signaling cascades are unknown. RESULTS: Here we report novel intraciliary calcium oscillations (ICOs) at the LRO that connect ciliary sensation of ciliary motility to downstream left-right signaling. Utilizing cilia-targeted genetically encoded calcium indicators in live zebrafish embryos, we show that ICOs depend on Pkd2 and are left-biased at the LRO in response to ciliary motility. Asymmetric ICOs occur with onset of LRO ciliary motility, thus representing the earliest known LR asymmetric molecular signal. Suppression of ICOs using a cilia-targeted calcium sink reveals that they are essential for LR development. CONCLUSIONS: These findings demonstrate that intraciliary calcium initiates LR development and identify cilia as a functional ion signaling compartment connecting ciliary motility and flow to molecular LR signaling.

A murine Zic3 transcript with a premature termination codon evades nonsense-mediated decay during axis formation.

The ZIC transcription factors are key mediators of embryonic development and ZIC3 is the gene most commonly associated with situs defects (heterotaxy) in humans. Half of patient ZIC3 mutations introduce a premature termination codon (PTC). In vivo, PTC-containing transcripts might be targeted for nonsense-mediated decay (NMD). NMD efficiency is known to vary greatly between transcripts, tissues and individuals and it is possible that differences in survival of PTC-containing transcripts partially explain the striking phenotypic variability that characterizes ZIC3-associated congenital defects. For example, the PTC-containing transcripts might encode a C-terminally truncated protein that retains partial function or that dominantly interferes with other ZIC family members. Here we describe the katun (Ka) mouse mutant, which harbours a mutation in the Zic3 gene that results in a PTC. At the time of axis formation there is no discernible decrease in this PTC-containing transcript in vivo, indicating that the mammalian Zic3 transcript is relatively insensitive to NMD, prompting the need to re-examine the molecular function of the truncated proteins predicted from human studies and to determine whether the N-terminal portion of ZIC3 possesses dominant-negative capabilities. A combination of in vitro studies and analysis of the Ka phenotype indicate that it is a null allele of Zic3 and that the N-terminal portion of ZIC3 does not encode a dominant-negative molecule. Heterotaxy in patients with PTC-containing ZIC3 transcripts probably arises due to loss of ZIC3 function alone.

Zic3 is required in the migrating primitive streak for node morphogenesis and left-right patterning.

In humans, loss-of-function mutations in ZIC3 cause isolated cardiovascular malformations and X-linked heterotaxy, a disorder with abnormal left-right asymmetry of organs. Zic3 null mice recapitulate the human heterotaxy phenotype but also have early gastrulation defects, axial patterning defects and neural tube defects complicating an assessment of the role of Zic3 in cardiac development. Zic3 is expressed ubiquitously during critical stages of left-right patterning but its later expression in the developing heart remains controversial and the molecular mechanism(s) by which it causes heterotaxy are unknown. To define the temporal and spatial requirements, for Zic3 in left-right patterning, we generated conditional Zic3 mice and Zic3-LacZ-BAC reporter mice. The latter provide compelling evidence that Zic3 is expressed in the mouse node and absent in the heart. Conditional deletion using T-Cre identifies a requirement for Zic3 in the primitive streak and migrating mesoderm for proper left-right patterning and cardiac development. In contrast, Zic3 is not required in heart progenitors or the cardiac compartment. In addition, the data demonstrate abnormal node morphogenesis in Zic3 null mice and identify similar node dysplasia when Zic3 was specifically deleted from the migrating mesoderm and primitive streak. These results define the temporal and spatial requirements for Zic3 in node morphogenesis, left-right patterning and cardiac development and suggest the possibility that a requirement for Zic3 in node ultrastructure underlies its role in heterotaxy and laterality disorders.

[Disorders of laterality and heterotaxy in the foetus]. / Fetale Lageanomalien und Heterotaxien.

Disorders of laterality and heterotaxy syndromes are rare diseases with an incidence of 1-1.5/10,000 live births. They are associated with numerous viscerocardiac anomalies and malformations. In particular, heterotaxy syndromes are associated with complex cardiac and extracardiac malformations that have an important impact on the prenatal and postnatal course. A prenatal differentiation between the 2 main variants of heterotaxy--left and right isomerism--is possible by assessment of cardiac rhythm, anomalies of caval veins and descending aorta and concomittant cardiac and extracardiac anomalies. An exact diagnosis is mandatory for adequate counselling of the parents and planning of postnatal care. Left isomerism has a high intrauterine mortality, caused by early atrioventricular block with subsequent cardiac failure and hydrops. In contrast, right isomerism has a high postnatal mortality due to the more complex type of cardiac defects and splenic disorders. The type of associated cardiac and extracardiac anomalies determines the postnatal morbidity and mortality. Polysplenia and asplenia may be associated with immunological disorders, that cannot be ruled out in the prenatal period, and further complicate the postnatal course.

How insights from cardiovascular developmental biology have impacted the care of infants and children with congenital heart disease.

To illustrate the impact developmental biology and genetics have already had on the clinical management of the million infants born worldwide each year with CHD, we have chosen three stories which have had particular relevance for pediatric cardiologists, cardiothoracic surgeons, cardiac anesthesiologists, and cardiac nurses. First, we show how Margaret Kirby's finding of the unexpected contribution of an ectodermal cell population - the cranial neural crest - to the aortic arch arteries and arterial pole of the embryonic avian heart provided a key impetus to the field of cardiovascular patterning. Recognition that a majority of patients affected by the neurocristopathy DiGeorge syndrome have a chromosome 22q11 deletion, have also spurred tremendous efforts to characterize the molecular mechanisms contributing to this pathology, assigning a major role to the transcription factor Tbx1. Second, synthesizing the work of the last two decades by many laboratories on a wide gamut of metazoans (invertebrates, tunicates, agnathans, teleosts, lungfish, amphibians, and amniotes), we review the >20 major modifications and additions to the ancient circulatory arrangement composed solely of a unicameral (one-chambered), contractile myocardial tube and a short proximal aorta. Two changes will be discussed in detail - the interposition of a second cardiac chamber in the circulation and the septation of the cardiac ventricle. By comparing the developmental genetic data of several model organisms, we can better understand the origin of the various components of the multicameral (multi-chambered) heart seen in humans. Third, Martina Brueckner's discovery that a faulty axonemal dynein was responsible for the phenotype of the iv/iv mouse (the first mammalian model of human heterotaxy) focused attention on the biology of cilia. We discuss how even the care of the complex cardiac and non-cardiac anomalies seen in heterotaxy syndrome, which have long seemed impervious to advancements in surgical and medical intensive care, may yet yield to strategies grounded in a better understanding of the cilium. The fact that all cardiac defects seen in patients with full-blown heterotaxy can also be seen in patients without obvious laterality defects hints at important roles for ciliary function not only in left-right axis specification but also in cardiovascular morphogenesis. These three developmental biology stories illustrate how the remaining unexplained mortality and morbidity of congenital heart disease can be solved.

Prenatal echocardiographic diagnosis of cardiac right/left axis and malpositions according to standardized Cordes technique.

OBJECTIVE: The aim of this study was to evaluate distinguishing the right/left side of the fetus, cardiac axis and position according to the standardized Cordes technique in 20 cases with cardiac malposition. METHODS: We studied retrospectively 1536 cases whose fetal echocardiographic examinations were performed between 1999 and 2006 in prenatal cardiology unit. Among these, cardiac malpositions were determined in 20 cases. The cardiac axis and position were determined according to the Cordes technique. All cases were followed-up by serial fetal echocardiograms until birth or intrauterine death occurred. In cases of intrauterine death, an autopsy was performed. After birth, physical and echocardiographic examinations were done and prenatal and postnatal diagnoses were compared. RESULTS: Of 1536 fetal echocardiograms performed, 144 revealed congenital heart diseases (9.4%), among these cases 20 were diagnosed with cardiac malposition. Of cases with cardiac malposition, 16 had congenital heart disease, and four had extracardiac malformation. There were six cases of isolated dextrocardia, three cases of situs inversus totalis, six cases of situs ambiguous, and one case of situs inversus with isolated levocardia. Of four cases with extracardiac malformations, two cases had mesoposition, one had dextroposition, and one had extreme levoposition. In six cases the autopsy findings were the same as that their prenatal echocardiographic findings. When postnatal echocardiographic results of the remaining cases with cardiac malposition due to congenital heart disease were compared with prenatal diagnoses, the same echocardiographic findings were verified. CONCLUSION: The fetal right/left axis must be determined correctly for the accurate diagnosis of cardiac malpositions. Therefore, we recommend that Cordes technique provides a simple and reliable determination of the fetal right/left axis and fetal situs.