Congenital heart diseases (CHDs) are the most common types of birth defects, affecting approximately 1% of live births and remaining the leading cause of mortality. CHD patients often show a higher incidence of heterotaxy syndrome. However, the exact aetiology of CHD and heterotaxy syndrome remains unclear. In this study, targeted sequencing and Sanger sequencing were performed to analyze the exonic regions of 37 primary ciliary dysfunction (PCD)- related candidate genes in 42 CHD patients with heterotaxy syndrome. Variants affecting protein-coding regions were filtered according to databases of known variants and predicted in silico using functional prediction program. Thirty-four potential disease-causing heterozygous variants in 11 genes were identified in the 19 CHD patients with heterotaxy syndrome (45.2%, 19/42). The DNAH11 gene showed the highest mutation rate (16.7%; 14 of 84 alleles) among the CHD patients with heterotaxy. Fisher's exact test revealed a significant association of DNAH11 variants with CHD and heterotaxy (P = 0.0001). In families, six different compound heterozygous variants of DNAH11 were validated in family 1-5031 (p.W802X/p.M282I), family 2-5045 (p.T3460K/p.G4425S), family 3-5065 (p.G447R/p.L1157R), family 4-5130 (p.I2262T/p.D3800H), family 5-5707 (p.S1823fs/p.F2759L/p.R4395X) and family 6-5062 (p.D3610V/p.I243V). These findings suggest that the DNAH11 variants are significantly associated with CHD and heterotaxy syndrome and that compound heterozygous DNAH11 variants may be the common genetic cause of the development of familial CHD and heterotaxy syndrome.

To characterize the diversity and prevalence of thoraco-abdominal abnormalities in Bardet-Biedl syndrome (BBS), a model ciliopathy for understanding the role of cilia in human health.

Heterotaxy (Htx) syndrome comprises a class of congenital disorders resulting from malformations in left-right body patterning. Approximately 90% of patients with heterotaxy have serious congenital heart diseases; as a result, the survival rate and outcomes of Htx patients are not satisfactory. However, the underlying etiology and mechanisms in the majority of Htx cases remain unknown. The aim of this study was to investigate the function of rare copy number variants (CNVs) in the pathogenesis of Htx.

Heterotaxy syndrome (HS) involves dysfunction of multiple systems resulting from abnormal left-right (LR) body patterning. Most HS patients present with complex congenital heart diseases (CHD), the disability and mortality of HS patients are extremely high. HS has great heterogeneity in phenotypes and genotypes, which have rendered gene discovery challenging. The aim of this study was to identify novel genes that underlie pathogenesis of HS patients with CHD. Whole exome sequencing was performed in 25 unrelated HS cases and 100 healthy controls; 19 nonsynonymous variants in 6 novel candidate genes (FLNA, ITGA1, PCNT, KIF7, GLI1, KMT2D) were identified. The functions of candidate genes were further analyzed in zebrafish model by CRISPR/Cas9 technique. Genome-editing was successfully introduced into the gene loci of flna, kmt2d and kif7, but the phenotypes were heterogenous. Disruption of each gene disturbed normal cardiac looping while kif7 knockout had a more prominent effect on liver budding and pitx2 expression. Our results revealed three potential HS pathogenic genes with probably different molecular mechanisms.

Heterotaxy syndrome (HTX) is characterized by left-right (LR) asymmetry disturbances associated with severe heart malformations. However, the exact genetic cause of HTX pathogenesis remains unclear. The aim of this study was to investigate the pathogenic mechanism underlying heterotaxy syndrome. Targeted next-generation sequencing (NGS) was performed for twenty-two candidate genes correlated with LR axis development in sixty-six HTX patients from unrelated families. Variants were filtered from databases and predicted in silico using prediction programs. A total of twenty-one potential disease-causing variants were identified in seven genes. Next, we used Sanger sequencing to confirm the identified variants in the family pedigree and found a novel hemizygous mutation (c.890G > T, p.C297F) in the ZIC3 gene in a male patient that was inherited from his mother, who was a carrier. The results of functional indicated that this ZIC3 mutation decreases transcriptional activity, affects the affinity of the GLI-binding site and results in aberrant cellular localization in transfected cells. Moreover, morpholino-knockdown experiments in zebrafish demonstrated that zic3 mutant mRNA failed to rescue the abnormal phenotype, suggesting a role for the novel ZIC3 mutation in heterotaxy syndrome.

Heterotaxy (HTX) is a rare condition of abnormal thoraco-abdominal organ arrangement across the left-right axis of the body. The pathogenesis of HTX includes a derangement of the complex signaling at the left-right organizer early in embryogenesis involving motile and non-motile cilia. It can be inherited as a single-gene disorder, a phenotypic feature of a known genetic syndrome or without any clear genetic etiology. Most patients with HTX have complex cardiovascular malformations requiring surgical intervention. Surgical risks are relatively high due to several serious comorbidities often seen in patients with HTX. Asplenia or functional hyposplenism significantly increase the risk for sepsis and therefore require antimicrobial prophylaxis and immediate medical attention with fever. Intestinal rotation abnormalities are common among patients with HTX, although volvulus is rare and surgical correction carries substantial risk. While routine screening for intestinal malrotation is not recommended, providers and families should promptly address symptoms concerning for volvulus and biliary atresia, another serious morbidity more common among patients with HTX. Many patients with HTX have chronic lung disease and should be screened for primary ciliary dyskinesia, a condition of respiratory cilia impairment leading to bronchiectasis. Mental health and neurodevelopmental conditions need to be carefully considered among this population of patients living with a substantial medical burden. Optimal care of children with HTX requires a cohesive team of primary care providers and experienced subspecialists collaborating to provide compassionate, standardized and evidence-based care. In this statement, subspecialty experts experienced in HTX care and research collaborated to provide expert- and evidence-based suggestions addressing the numerous medical issues affecting children living with HTX.

Primary ciliary dyskinesia (PCD) is a clinically heterogeneous group of autosomal or, less frequently, X-chromosomal recessive inheritance syndrome of motile cilia dysfunction characterized by neonatal respiratory distress, oto-sino-pulmonary disease, infertility and situs inversus. Recently, type 43 PCD (CILD43, OMIM#618699) was established by autosomal-dominant loss-of-function mutations identified in Forkhead box J1 (FOXJ1). However, the functional validation of FOXJ1 mutations in humans and mice has not been fully performed. Here we studied a three-generation family with heterotaxy and proband with complex congenital heart disease (CHD).

Heterotaxy syndrome (HTX) is caused by aberrant left-right patterning early in embryonic development, which results in abnormal positioning and morphology of the thoracic and abdominal organs. Currently, genetic testing discerns the underlying genetic cause in less than 20% of sporadic HTX cases, indicating that genetic pathogenesis remains poorly understood. In this study, we aim to garner a deeper understanding of the genetic factors of this disease by documenting the effect of different matrix metalloproteinase 21 (MMP21) variants on disease occurrence and pathogenesis.

Primary ciliary dyskinesia (PCD), a rare genetic disorder, is mostly caused by defects in more than 40 known cilia structure-related genes. However, in approximately 20-35% of patients, it is caused by unknown genetic factors, and the inherited pathogenic factors are difficult to confirm. Kartagener syndrome (KTS) is a subtype of PCD associated with situs inversus, presenting more complex genetic heterogeneity. The aim of this study was to identify pathogenic mutations of candidate genes in Chinese patients with KTS and investigate the activation of the heterotaxy-related NOTCH pathway. Whole-exome sequencing was conducted in five patients with KTS. Pathogenic variants were identified using bioinformatics analysis. Candidate variants were validated by Sanger sequencing. The expression of the NOTCH pathway target genes was detected in patients with KTS. We identified 10 KTS-associated variants in six causative genes, namely, CCDC40, DNAH1, DNAH5, DNAH11, DNAI1, and LRRC6. Only one homozygote mutation was identified in LRRC6 (c.64dupT). Compound heterozygous mutations were found in DNAH1 and DNAH5. Six novel mutations were identified in four genes. Further analyses showed that the NOTCH pathway might be activated in patients with KTS. Overall, our study showed that compound heterozygous mutations widely exist in Chinese KTS patients. Our results demonstrated that the activation of the NOTCH pathway might play a role in the situs inversus pathogenicity of KTS. These findings highlight that Kartagener syndrome might be a complex genetic heterogeneous disorder mediated by heterozygous mutations in multiple PCD- or cilia-related genes.

Atrioventricular septal defects (AVSD) are common cardiovascular malformations, occurring in 3.5/10,000 births. Although frequently associated with trisomy 21, autosomal dominant AVSD has also been described. Recently we identified and characterized the cell adhesion molecule CRELD1 (previously known as "cirrin") as a candidate gene for the AVSD2 locus mapping to chromosome 3p25. Analysis of the CRELD1 gene from individuals with non-trisomy 21-associated AVSD identified heterozygous missense mutations in nearly 6% of this population, including mutations in isolated AVSD and AVSD associated with heterotaxy syndrome. CRELD1 is the first human gene to be implicated in the pathogenesis of isolated AVSD and AVSD in the context of heterotaxy, which provides an important step in unraveling the pathogenesis of AVSD.

The ciliary kinase NEK8 plays a critical role in situs determination and cystic kidney disease, yet its exact function remains unknown. In this study, we identify ANKS6 as a target and activator of NEK8. ANKS6 requires NEK8 for localizing to the ciliary inversin compartment (IC) and activates NEK8 by binding to its kinase domain. Here we demonstrate the functional importance of this interaction through the analysis of two novel mouse mutations, Anks6(Streaker) and Nek8(Roc). Both display heterotaxy, cardiopulmonary malformations and cystic kidneys, a syndrome also characteristic of mutations in Invs and Nphp3, the other known components of the IC. The Anks6(Strkr) mutation decreases ANKS6 interaction with NEK8, precluding NEK8 activation. The Nek8(Roc) mutation inactivates NEK8 kinase function while preserving ANKS6 localization to the IC. Together, these data reveal the crucial role of NEK8 kinase activation within the IC, promoting proper left-right patterning, cardiopulmonary development and renal morphogenesis.

Most isolated congenital heart defects are thought to be sporadic and are often ascribed to multifactorial mechanisms with poorly understood genetics. Total Anomalous Pulmonary Venous Return (TAPVR) occurs in 1 in 15,000 live-born infants and occurs either in isolation or as part of a syndrome involving aberrant left-right development. Previously, we reported causative links between TAVPR and the PDGFRA gene. TAPVR has also been linked to the ANKRD1/CARP genes. However, these genes only explain a small fraction of the heritability of the condition. By examination of phased single nucleotide polymorphism genotype data from 5 distantly related TAPVR patients we identified a single 25 cM shared, Identical by Descent genomic segment on the short arm of chromosome 12 shared by 3 of the patients and their obligate-carrier parents. Whole genome sequence (WGS) analysis identified a non-synonymous variant within the shared segment in the retinol binding protein 5 (RBP5) gene. The RBP5 variant is predicted to be deleterious and is overrepresented in the TAPVR population. Gene expression and functional analysis of the zebrafish orthologue, rbp7, supports the notion that RBP5 is a TAPVR susceptibility gene. Additional sequence analysis also uncovered deleterious variants in genes associated with retinoic acid signaling, including NODAL and retinol dehydrogenase 10. These data indicate that genetic variation in the retinoic acid signaling pathway confers, in part, susceptibility to TAPVR.

The etiology of congenital heart defects (CHDs), which are among the most common human birth defects, is poorly understood because of its complex genetic architecture. Here, we show that two genes implicated in CHDs, Megf8 and Mgrn1, interact genetically and biochemically to regulate the strength of Hedgehog signaling in target cells. MEGF8, a transmembrane protein, and MGRN1, a RING superfamily E3 ligase, assemble to form a receptor-like ubiquitin ligase complex that catalyzes the ubiquitination and degradation of the Hedgehog pathway transducer Smoothened. Homozygous Megf8 and Mgrn1 mutations increased Smoothened abundance and elevated sensitivity to Hedgehog ligands. While mice heterozygous for loss-of-function Megf8 or Mgrn1 mutations were normal, double heterozygous embryos exhibited an incompletely penetrant syndrome of CHDs with heterotaxy. Thus, genetic interactions can arise from biochemical mechanisms that calibrate morphogen signaling strength, a conclusion broadly relevant for the many human diseases in which oligogenic inheritance is emerging as a mechanism for heritability.

Laterality defects (LDs) or asymmetrically positioned organs are a group of rare developmental disorders caused by environmental and/or genetic factors. However, the exact molecular pathophysiology of LD is not yet fully characterised. In this context, studying Arab population presents an ideal opportunity to discover the novel molecular basis of diseases owing to the high rate of consanguinity and genetic disorders. Therefore, in the present study, we studied the molecular basis of LD in Arab patients, using next-generation sequencing method. We discovered an extremely rare novel missense variant in MYO1D gene (Pro765Ser) presenting with visceral heterotaxy and left isomerism with polysplenia syndrome. The proband in this index family has inherited this homozygous variant from her heterozygous parents following the autosomal recessive pattern. This is the first report to show MYO1D genetic variant causing left-right axis defects in humans, besides previous known evidence from zebrafish, frog and Drosophila models. Moreover, our multilevel bioinformatics-based structural (protein variant structural modelling, divergence, and stability) analysis has suggested that Ser765 causes minor structural drifts and stability changes, potentially affecting the biophysical and functional properties of MYO1D protein like calmodulin binding and microfilament motor activities. Functional bioinformatics analysis has shown that MYO1D is ubiquitously expressed across several human tissues and is reported to induce severe phenotypes in knockout mouse models. In conclusion, our findings show the expanded genetic spectrum of LD, which could potentially pave way for the novel drug target identification and development of personalised medicine for high-risk families.

Defects in laterality pattern can result in abnormal positioning of the internal organs during the early stages of embryogenesis, as manifested in heterotaxy syndrome and situs inversus, while laterality defects account for 3~7% of all congenital heart defects (CHDs). However, the pathogenic mechanism underlying most laterality defects remains unknown. In this study, we recruited 70 laterality defect patients with CHDs to identify candidate disease genes by exome sequencing. We then evaluated rare, loss-of-function (LOF) variants, identifying candidates by referring to previous literature. We chose TRIP11, DNHD1, CFAP74, and EGR4 as candidates from 776 LOF variants that met the initial screening criteria. After the variants-to-gene mapping, we performed function research on these candidate genes. The expression patterns and functions of these four candidate genes were studied by whole-mount in situ hybridization, gene knockdown, and gene rescue methods in zebrafish models. Among the four genes, trip11, dnhd1, and cfap74 morphant zebrafish displayed abnormalities in both cardiac looping and expression patterns of early signaling molecules, suggesting that these genes play important roles in the establishment of laterality patterns. Furthermore, we performed immunostaining and high-speed cilia video microscopy to investigate Kupffer's vesicle organogenesis and ciliogenesis of morphant zebrafish. Impairments of Kupffer's vesicle organogenesis or ciliogenesis were found in trip11, dnhd1, and cfap74 morphant zebrafish, which revealed the possible pathogenic mechanism of their LOF variants in laterality defects. These results highlight the importance of rare, LOF variants in identifying disease-related genes and identifying new roles for TRIP11, DNHD1, and CFAP74 in left-right patterning. Additionally, these findings are consistent with the complex genetics of laterality defects.

A whole-genome association study of 4631 progeny-tested Nordic Red dairy cattle bulls using imputed next-generation sequencing data revealed a major quantitative trait locus (QTL) that affects birth index (BI) on Bos taurus autosome (BTA) 23. We analyzed this QTL to identify which of the component traits of BI are affected and understand its molecular basis.