Whole-exome sequencing reveals a combination of extremely rare single-nucleotide polymorphism of DNAH9 and RSPH1 genes in a Japanese fetus with situs viscerum inversus.

Randomization of left-right body asymmetry, situs viscerum inversus (heterotaxy), is commonly associated with primary ciliary dyskinesia (PCD) resulting from an abnormal ciliary structure, with approximately 50% of PCD patients exhibiting organ laterality defects. I herein report an intrauterine fetal death case, in which an autopsy revealed two lobes of the bilateral lungs as well as heterotaxy of abdominal organs (right-sided spleen and inversion of the alimentary and biliary organs). Whole-exome sequencing (WES) identified a heterozygous single-nucleotide change (c.12775T>C) in exon 68 of the DNAH9 gene, which is a rare single-nucleotide polymorphism (SNP) of rs746081639 and results in the amino acid change of p.C4259R. WES also identified a rare SNP of rs763089682 (c.121G>A) in the RSPH1 gene that causes a heterozygous amino acid alteration of p.G41R. The frequencies of both SNPs, C in rs746081639 and A in rs763089682, are 0.00000824, and a polyphen-2 analysis predicted these amino acid changes to be probably damaging, with a score of 1.000. The combination of extremely rare SNPs in DNAH9 and RSPH1 genes might have been the possible mechanism underlying the development of the laterality defect in the present case.

Phenotypic features of ciliary dyskinesia among patients with congenital cardiovascular malformations.

BACKGROUND: Cilia are cell membrane-bound organelles responsible for airway mucus clearance, establishment of left-right organ asymmetry, cardiogenesis, and many other functions in utero. Phenotypic features suggestive of respiratory ciliary dyskinesia among patients with heterotaxy syndrome, defined as complex cardiovascular malformations (CVM) and situs ambiguus (SA), has not been adequately explored. OBJECTIVES: We hypothesized that there is a greater incidence of phenotypic features consistent with ciliary dyskinesia among patients with heterotaxy syndrome compared to patients with other CVM and laterality defects without heterotaxy syndrome. METHODS: Thirty six subjects were identified by medical record search and divided into four groups based on situs status and type of CVM as follows: SA and complex CVM (group 1); SA and simple CVM (group 2); situs solitus and complex CVM (group 3); and situs solitus and simple CVM (group 4). Phenotype was assessed with a clinical questionnaire, nasal nitric oxide (NO) level, and pulmonary function testing. Those with complex CVM underwent additional testing for variants in genes involved in ciliary structure and function. RESULTS: The mean nasal NO level was significantly lower among all subjects with complex CVM regardless of situs anomalies (groups 1 and 3). There was no significant difference in respiratory symptoms or lung function among the four groups. No bi-allelic genetic mutations were detected among patients with complex CVM. CONCLUSIONS: This study identified a relatively lower mean nasal NO level, suggestive of relative ciliary dyskinesia, among subjects with complex CVM. Pulmonary function and clinical symptoms did not reflect significant pulmonary disease among those with complex CVM.

Genetic basis of human left-right asymmetry disorders.

Humans and other vertebrates exhibit left-right (LR) asymmetric arrangement of the internal organs, and failure to establish normal LR asymmetry leads to internal laterality disorders, including situs inversus and heterotaxy. Situs inversus is complete mirror-imaged arrangement of the internal organs along LR axis, whereas heterotaxy is abnormal arrangement of the internal thoraco-abdominal organs across LR axis of the body, most of which are associated with complex cardiovascular malformations. Both disorders are genetically heterogeneous with reduced penetrance, presumably because of monogenic, polygenic or multifactorial causes. Research in genetics of LR asymmetry disorders has been extremely prolific over the past 17 years, and a series of loci and disease genes involved in situs inversus and heterotaxy have been described. The review highlights the classification, chromosomal abnormalities, pathogenic genes and the possible mechanism of human LR asymmetry disorders.

Nephrocystin-3 is required for ciliary function in zebrafish embryos.

Nephronophthisis (NPHP) is the most frequent genetic cause of end-stage renal failure in the first three decades of life. It is characterized primarily by renal cysts with extrarenal involvements of the eye and brain. Ten recessive genes responsible for NPHP have been identified by positional cloning. This discovery supported a unifying theory of renal cystic disease, which states that all proteins mutated in cystic kidney diseases of human, mice, or zebrafish are expressed in primary cilia of renal epithelial cells. Mutations in nephrocystin-3 (NPHP3) are the cause of human nephronophthisis type 3 and polycystic kidney disease (pcy) mouse mutants. To study the functional role of NPHP3 in normal embryonic development and in the pathogenesis of cystic kidney disease, we characterized the zebrafish ortholog nphp3 by morpholino oligo (MO)-mediated knockdown. When nphp3 function was suppressed by either of the two MOs blocking the translation of the protein or the splicing of mRNA, zebrafish embryos displayed hydrocephalus and pronephric cysts. Knockdown of nphp3 also led to situs inversus phenotypes due to defective cilia at Kupffer's vesicle. We showed that nphp3 genetically interacts with nphp2/inversin and human NPHP3 localizes to primary cilia in Madin-Darby canine kidney cells. Like nphp2/inversin, nphp3 knockdown affected morphogenic cell movement during gastrulation, suggesting nphp3 is essential to regulate convergent extension. Thus nphp3, cooperating with nphp2/inversin, plays an essential role related to ciliary function, and the knockdown provides an animal model that may be used for studies of the pathogenesis and therapy for this disease.

Consistent left-right asymmetry cannot be established by late organizers in Xenopus unless the late organizer is a conjoined twin.

How embryos consistently orient asymmetries of the left-right (LR) axis is an intriguing question, as no macroscopic environmental cues reliably distinguish left from right. Especially unclear are the events coordinating LR patterning with the establishment of the dorsoventral (DV) axes and midline determination in early embryos. In frog embryos, consistent physiological and molecular asymmetries manifest by the second cell cleavage; however, models based on extracellular fluid flow at the node predict correct de novo asymmetry orientation during neurulation. We addressed these issues in Xenopus embryos by manipulating the timing and location of dorsal organizer induction: the primary dorsal organizer was ablated by UV irradiation, and a new organizer was induced at various locations, either early, by mechanical rotation, or late, by injection of lithium chloride (at 32 cells) or of the transcription factor XSiamois (which functions after mid-blastula transition). These embryos were then analyzed for the position of three asymmetric organs. Whereas organizers rescued before cleavage properly oriented the LR axis 90% of the time, organizers induced in any position at any time after the 32-cell stage exhibited randomized laterality. Late organizers were unable to correctly orient the LR axis even when placed back in their endogenous location. Strikingly, conjoined twins produced by late induction of ectopic organizers did have normal asymmetry. These data reveal that although correct LR orientation must occur no later than early cleavage stages in singleton embryos, a novel instructive influence from an early organizer can impose normal asymmetry upon late organizers in the same cell field.

Advanced cancer with situs inversus totalis associated with KIF3 complex deficiency: report of two cases.

Situs inversus totalis (SIT) is a relatively rare congenital anomaly, occurring at an incidence of 1 in 10 000-50 000 live births. Although there are some case reports of SIT with the presence of cancer, there are few reports on the relationship between SIT and cancer. However, the recent phylogenetic investigations of this condition suggest that this may be linked to the development and progression of cancer on the molecular level. The key elements are one of the intracellular motor proteins, the KIF3 complex, and the cell-adhesion factors N-cadherin and beta-catenin. We herein present the cases of advanced gastric cancer and lung cancer with SIT, and review the relationship between SIT and the development and progression of cancer.

The subcellular localization of TRPP2 modulates its function.

TRPP2, also known as polycystin-2, is a calcium permeable nonselective cation channel that is mutated in autosomal dominant polycystic kidney disease but has also been implicated in the regulation of cardiac development, renal tubular differentiation, and left-to-right (L-R) axis determination. For obtaining further insight into how TRPP2 exerts tissue-specific functions, this study took advantage of PACS-dependent trafficking of TRPP2 in zebrafish larvae. PACS proteins recognize an acidic cluster within the carboxy-terminal domain of TRPP2 that undergoes phosphorylation and mediate retrieval of TRPP2 to the Golgi and endoplasmic reticulum (ER). The interaction of human TRPP2 with PACS proteins can be inhibited by a Ser812Ala mutation (TRPP2(S812A)), thereby allowing TRPP2 to reach other subcellular compartments, and enhanced by a Ser812Asp mutation (TRPP2(S812D)), thereby trapping TRPP2 in the ER. It was found that the TRPP2(S812A) mutant rescued cyst formation of TRPP2-deficient zebrafish larvae to the same degree as wild-type TRPP2, whereas the TRPP2(S812D) mutant was significantly more effective in normalizing the distorted body axis of TRPP2-deficient fish. Surprisingly, the TRPP2(S812D) mutant rescued the abnormalities of L-R asymmetry more effectively than either wild-type or TRPP2(S812A), suggesting that the ER localization of TRPP2 plays an important role in the development of normal L-R asymmetry. Taken together, these findings support the hypothesis that TRPP2 assumes distinct subcellular localizations to exert tissue-specific functions.

Left-right lineage analysis of the embryonic Xenopus heart reveals a novel framework linking congenital cardiac defects and laterality disease.

The significant morbidity and mortality associated with laterality disease almost always are attributed to complex congenital heart defects (CHDs), reflecting the extreme susceptibility of the developing heart to disturbances in the left-right (LR) body plan. To determine how LR positional information becomes ;translated' into anatomical asymmetry, left versus right side cardiomyocyte cell lineages were traced in normal and laterality defective embryos of the frog, Xenopus laevis. In normal embryos, myocytes in some regions of the heart were derived consistently from a unilateral lineage, whereas other regions were derived consistently from both left and right side lineages. However, in heterotaxic embryos experimentally induced by ectopic activation or attenuation of ALK4 signaling, hearts contained variable LR cell composition, not only compared with controls but also compared with hearts from other heterotaxic embryos. In most cases, LR cell lineage defects were associated with abnormal cardiac morphology and were preceded by abnormal Pitx2c expression in the lateral plate mesoderm. In situs inversus embryos there was a mirror image reversal in Pitx2c expression and LR lineage composition. Surprisingly, most of the embryos that failed to develop heterotaxy or situs inversus in response to misregulated ALK4 signaling nevertheless had altered Pitx2c expression, abnormal cardiomyocyte LR lineage composition and abnormal heart structure, demonstrating that cardiac laterality defects can occur even in instances of otherwise normal body situs. These results indicate that: (1) different regions of the heart contain distinct LR myocyte compositions; (2) LR cardiomyocyte lineages and Pitx2c expression are altered in laterality defective embryos; and (3) abnormal LR cardiac lineage composition frequently is associated with cardiac malformations. We propose that proper LR cell composition is necessary for normal morphogenesis, and that misallocated LR cell lineages may be causatively linked with CHDs that are present in heterotaxic individuals, as well as some 'isolated' CHDs that are found in individuals lacking overt features of laterality disease.

Roles of the Foxj1 and Inv genes in the left-right determination of internal organs in mice.

In Foxj1 knockout mice, half show situs solitus while the other half show situs inversus, which means a random determination of the left-right axis. In contrast, the inv mutant mice show a mirror-image configuration of the internal organs, which means a reversal of the left-right axis. Although these two mutant mice have primary cilia on the nodal cells, their phenotypes are different in laterality determination. We thus made Foxj1/inv double mutant mice and analyzed their phenotype. We found the phenotypes of Foxj1/inv double mutant mice to be more similar to those of the Foxj1 mutant mice than those of the inv mutant mice. We also found right pulmonary isomerism to be a major phenotype of the Foxj1 mutant mice and the Foxj1/inv double mutant mice, which is likely due to the absence of the Pitx2 expression at both lateral plate mesoderms. These results indicate that a random signal of laterality (Foxj1) is dominant over the reversal signal of laterality (Inv).

Left-right lineage analysis of AV cushion tissue in normal and laterality defective Xenopus hearts.

The majority of complex congenital heart defects occur in individuals who are afflicted by laterality disease. We hypothesize that the prevalence of valvuloseptal defects in this population is due to defective left-right patterning of the embryonic atrioventricular (AV) canal cushions, which are the progenitor tissue for valve and septal structures in the mature heart. Using embryos of the frog Xenopus laevis, this hypothesis was tested by performing left-right lineage analysis of myocytes and cushion mesenchyme cells of the superior and inferior cushion regions of the AV canal. Lineage analyses were conducted in both wild-type and laterality mutant embryos experimentally induced by misexpression of ALK4, a type I TGF-beta receptor previously shown to modulate left-right axis determination in Xenopus. We find that abnormalities in overall amount and left-right cell lineage composition are present in a majority of ALK4-induced laterality mutant embryos and that much variation in the nature of these abnormalities exists in embryos that exhibit the same overall body situs. We propose that these two parameters of cushion tissue formation-amount and left-right lineage origin-are important for normal processes of valvuloseptal morphogenesis and that defective allocation of cells in the AV canal might be causatively linked to the high incidence of valvuloseptal defects associated with laterality disease.

Reversed bodies, reversed brains, and (some) reversed behaviors: of zebrafish and men.

Although zebrafish with situs inversus show complete reversal of normal visceral and cerebral asymmetries, they show left-right reversal of only some behaviors, with others continuing to show species-typical lateralization. The implication is that, as in humans, there are at least two independent mechanisms for generating asymmetry.

Intraflagellar transport and cilia-dependent diseases.

Intraflagellar transport involves the movement of large protein particles along ciliary microtubules and is required for the assembly and maintenance of eukaryotic cilia and flagella. Intraflagellar-transport defects in the mouse cause a range of diseases including polycystic kidney disease, retinal degeneration and the laterality abnormality situs inversus, highlighting the important role that motile, sensory and primary cilia play in vertebrates.

alpha(1)-Adrenergic stimulation perturbs the left-right asymmetric expression pattern of nodal during rat embryogenesis.

BACKGROUND: Normal development of the left/right (L/R) body axis leads to the characteristic sidedness of asymmetric body structures, e.g., the left-sided heart. Several genes are now known to be expressed with L/R asymmetry during embryogenesis, including nodal, a member of the transforming growth factor-beta (TGF-beta) family. Mutations or experimental treatments that affect L/R development, such as those that cause situs inversus (reversal of the sidedness of asymmetric body structures), have been shown to alter or abolish nodal's asymmetric expression. METHODS: In the present study, we examined the effects on nodal expression of alpha(1)-adrenergic stimulation, known to cause a 50% incidence of situs inversus in rat embryos grown in culture, using reverse transcription-polymerase chain reaction assay and whole-mount in situ hybridization assay. RESULTS: In embryos cultured with phenylephrine, an alpha(1)-adrenergic agonist, nodal's normal asymmetric expression only in the left lateral plate mesoderm was altered. In some treated embryos, nodal expression was detected in either the left or right lateral plate mesoderm. However, most treated embryos lacked lateral plate mesoderm expression. In addition, the embryos that did show expression were at a later stage than when nodal expression is normally found. CONCLUSIONS: Our results demonstrate that alpha(1)-adrenergic stimulation delays the onset and perturbs the normal asymmetric pattern of nodal expression. Either of these effects might contribute to situs inversus.

Ciliogenesis and left-right axis defects in forkhead factor HFH-4-null mice.

Cilia have been classified as sensory or motile types on the basis of functional and structural characteristics; however, factors important for regulation of assembly of different cilia types are not well understood. Hepatocyte nuclear factor-3/forkhead homologue 4 (HFH-4) is a winged helix/forkhead transcription factor expressed in ciliated cells of the respiratory tract, oviduct, and ependyma in late development through adulthood. Targeted deletion of the Hfh4 gene resulted in defective ciliogenesis in airway epithelial cells and randomized left-right asymmetry so that half the mice had situs inversus. In HFH-4-null mice, classic motile type cilia with a 9 + 2 microtubule ultrastructure were absent in epithelial cells, including those in the airways. In other organs, sensory cilia with a 9 + 0 microtubule pattern, such as those on olfactory neuroepithelial cells, were present. Ultrastructural analysis of mutant cells with absent 9 + 2 cilia demonstrated that defective ciliogenesis was due to abnormal centriole migration and/or apical membrane docking, suggesting that HFH-4 functions to direct basal body positioning or anchoring. Evaluation of wild-type embryos at gestational days 7.0 to 7.5 revealed Hfh4 expression in embryonic node cells that have monocilium, consistent with a function for this factor at the node in early determination of left- right axis. Analysis of the node of HFH-4 mutant embryos revealed that, in contrast to absent airway cilia, node cilia were present. These observations indicate that there are independent regulatory pathways for node ciliogenesis compared with 9 + 2 type ciliogenesis in airways, and support a central role for HFH-4 in ciliogenesis and left-right axis formation.

Adrenergic neurotransmitters and calcium ionophore-induced situs inversus viscerum in Xenopus laevis embryos.

Xenopus laevis embryos at the blastula-early tail bud stage were exposed to norepinephrine or octopamine dissolved in culture saline until they reached the larval stage. The left-right asymmetry of the heart and gut was then examined. We found that these adrenergic neurotransmitters induced situs inversus in the heart and/or gut in up to 35% of tested neurula embryos. Norepinephrine-induced situs inversus was blocked by the alpha-1 adrenergic antagonist prazosin. Furthermore, A23187, a calcium ionophore, also increased the incidence of situs inversus up to 54% when late-neurula embryos were exposed to the solution. A23187 treatment initiated before neural groove formation was less effective. The incidence of situs inversus induced by these reagents decreased towards the control level (2.2%, 25 untreated embryos out of 1127 embryos in total) in embryos past the stage of neural tube closure. In the present experiments we obtained 22 gut-only situs inversus embryos having an inverted gut and a normal heart. In contrast, such embryos were not observed among the 1127 untreated embryos. An adrenergic signal mediated by an increase in intracellular free calcium may be involved in the asymmetrical visceral morphogenesis of Xenopus embryos.