PRKACA-related, atrial defects-polydactyly-multiple congenital malformation syndrome in an Indian patient.

PRKACA-related, atrial defects-polydactyly-multiple congenital malformation syndrome is a recently described skeletal ciliopathy, which is caused by disease-causing variants in PRKACA. The primary phenotypic description includes atrial septal defects, and limb anomalies including polydactyly and short limbs. To date, only four molecularly proven patients have been reported in the literature with a recurrent variant, c.409G>A p.Gly137Arg in PRKACA. In this study, we report the fifth affected individual with the same variant and review the clinical features and radiographic findings of this rare syndrome.

Molecular Pathways and Animal Models of Atrial Septal Defect.

The relative simplicity of the clinical presentation and management of an atrial septal defect belies the complexity of the developmental pathogenesis. Here, we describe the anatomic development of the atrial septum and the venous return to the atrial chambers. Experimental models suggest how mutations and naturally occurring genetic variation could affect developmental steps to cause a defect within the oval fossa, the so-called secundum defect, or other interatrial communications, such as the sinus venosus defect or ostium primum defect.

Molecular Pathways and Animal Models of Atrioventricular Septal Defect.

The development of a fully functional four-chambered heart is critically dependent on the correct formation of the structures that separate the atrial and ventricular chambers. Perturbation of this process typically results in defects that allow mixing of oxygenated and deoxygenated blood. Atrioventricular septal defects (AVSD) form a class of congenital heart malformations that are characterized by the presence of a primary atrial septal defect (pASD), a common atrioventricular valve (cAVV), and frequently also a ventricular septal defect (VSD). While AVSD were historically considered to result from failure of the endocardial atrioventricular cushions to properly develop and fuse, more recent studies have determined that inhibition of the development of other components of the atrioventricular mesenchymal complex can lead to AVSDs as well. The role of the dorsal mesenchymal protrusion (DMP) in AVSD pathogenesis has been well-documented in studies using animal models for AVSDs, and in addition, preliminary data suggest that the mesenchymal cap situated on the leading edge of the primary atrial septum may be involved in certain situations as well. In this chapter, we review what is currently known about the molecular mechanisms and animal models that are associated with the pathogenesis of AVSD.

Tbx5 inhibits hedgehog signaling in determination of digit identity.

Dominant TBX5 mutation causes Holt-Oram syndrome (HOS), which is characterized by limb defects in humans, but the underlying mechanistic basis is unclear. We used a mouse model with Tbx5 conditional knockdown in Hh-receiving cells (marked by Gli1+) during E8 to E10.5, a previously established model to study atrial septum defects, which displayed polydactyly or hypodactyly. The results suggested that Tbx5 is required for digit identity in a subset of limb mesenchymal cells. Specifically, Tbx5 deletion in this cell population decreased cell apoptosis and increased the proliferation of handplate mesenchymal cells. Furthermore, Tbx5 was found to negatively regulate the Hh-signaling activity through transcriptional regulation of Ptch1, a known Hh-signaling repressor. Repression of Hh-signaling through Smo co-mutation in Tbx5 heterozygotes rescued the limb defects, thus placing Tbx5 upstream of Hh-signaling in limb defects. This work reveals an important missing component necessary for understanding not only limb development but also the molecular and genetic mechanisms underlying HOS.

Identification of a novel TBX5 c.755 + 1 G > A variant and related pathogenesis in a family with Holt-Oram syndrome.

The proband with congenital heart disease and abnormal thumb was clinically diagnosed as Holt-Oram syndrome (HOS). A novel variant, T-box transcription factor 5 (TBX5) c.755 + 1 G > A, was identified in the proband via whole exome sequencing and validated using Sanger sequencing. Pedigree analysis and clinical examinations revealed three/seven individuals over three generations within the family, with features suggestive of HOS. Deep amplicon sequencing confirmed that the allele frequencies of the novel variant in the proband (III-1), her brother (III-2), and her mother (II-2) were 50%, 48.3%, and 38.1%, respectively, indicating that III-1 and III-2 harbored heterozygous variants, while II-2 harbored mosaic heterozygous variants. The minigene splicing assay showed that the novel variant affected the normal splicing of exon 7, resulting in the production of abnormal TBX5 transcripts. Reverse transcription-quantitative polymerase chain reaction and western blot analyses revealed that the novel variant upregulated TBX5 expression at the transcriptional and translational levels. Nuclear localization assay demonstrated impaired nuclear localization of the mutant TBX5. Cell viability assay revealed the inhibition of cell activity by the mutant TBX5. Our findings indicate that the novel variant was potentially induced HOS, probably by causing aberrant splicing, reducing the enrichment of nuclear TBX5 protein, and inhibiting cellular proliferation.

Functional analysis of two novel TBX5 variants present in individuals with Holt-Oram syndrome with different clinical manifestations.

Holt-Oram syndrome (HOS) is a rare disorder characterized by cardiac and upper-limb defects. Pathogenic variants in TBX5-a gene encoding a transcription factor important for heart and skeletal development-are the only known cause of HOS. Here, we present the identification and functional analysis of two novel TBX5 pathogenic variants found in two individuals with HOS presenting distinct phenotypes. The individual with the c.905delA variant has a severe cardiac phenotype but mild skeletal defects, unlike the individual with the c.246_249delGATG variant who has no cardiac problems but severe upper limbs malformations, including phocomelia. Both frameshift variants, c.246_249delGATG and c.905delA, generate mRNAs harbouring premature stop codons which, if not degraded by nonsense mediated decay, will lead to the production of shorter TBX5 proteins, p.Gln302Argfs*92 and p.Met83Phefs*6, respectively. Immunocytochemistry results suggest that both mutated proteins are produced and furthermore, like the wild-type protein, p.Gln302Argfs*92 mutant appears to be mainly localized in the nucleus, in contrast with p.Met83Phefs*6 mutant that displays a higher level of cytoplasmic localization. In addition, luciferase activity analysis revealed that none of the TBX5 mutants are capable of transactivating the NPPA promoter. In conclusion, our results provide evidence that both pathogenic variants cause a severe TBX5 loss-of-function, dramatically reducing its biological activity. The absence of cardiac problems in the individual with the p.Met83Phefs*6 variant supports the existence of other mechanisms/genes underlying the pathogenesis of HOS and/or the existence of an age-related delay in the development of a more serious cardiac phenotype. Further studies are required to understand the differential effects observed in the phenotypes of both individuals.

Biallelic VPS35L pathogenic variants cause 3C/Ritscher-Schinzel-like syndrome through dysfunction of retriever complex.

BACKGROUND: 3C/Ritscher-Schinzel syndrome is characterised by congenital cranio-cerebello-cardiac dysplasia, where CCDC22 and WASHC5 are accepted as the causative genes. In combination with the retromer or retriever complex, these genes play a role in endosomal membrane protein recycling. We aimed to identify the gene abnormality responsible for the pathogenicity in siblings with a 3C/Ritscher-Schinzel-like syndrome, displaying cranio-cerebello-cardiac dysplasia, coloboma, microphthalmia, chondrodysplasia punctata and complicated skeletal malformation. METHODS: Exome sequencing was performed to identify pathogenic variants. Cellular biological analyses and generation of knockout mice were carried out to elucidate the gene function and pathophysiological significance of the identified variants. RESULTS: We identified compound heterozygous pathogenic variants (c.1097dup; p.Cys366Trpfs*28 and c.2755G>A; p.Ala919Thr) in the VPS35L gene, which encodes a core protein of the retriever complex. The identified missense variant lacked the ability to form the retriever complex, and the frameshift variant induced non-sense-mediated mRNA decay, thereby confirming biallelic loss of function of VPS35L. In addition, VPS35L knockout cells showed decreased autophagic function in nutrient-rich and starvation conditions, as well as following treatment with Torin 1. We also generated Vps35l-/- mice and demonstrated that they were embryonic lethal at an early stage, between E7.5 and E10.5. CONCLUSIONS: Our results suggest that biallelic loss-of-function variants in VPS35L underlies 3C/Ritscher-Schinzel-like syndrome. Furthermore, VPS35L is necessary for autophagic function and essential for early embryonic development. The data presented here provide a new insight into the critical role of the retriever complex in fetal development.

Deployed Dimensions of the GORE® CARDIOFORM ASD Occluder as Function of Defect Size.

The GORE® CARDIOFORM ASD occluder (ASDO) is approved for closure of ASDs up to 35 mm diameter. With an adaptable central waist, each device size is suitable over a range of defect diameters. Understanding deployed dimensions across various defect sizes will assist operators. Therefore, this study investigates the deployed dimensions of the ASDO as a function of defect size. A 2-mm-thick ASD model with circular defects ranging from 5 to 35 mm was 3D printed. Diameter, width, and left-right disc diameter were measured by fluoroscopy after ASDO devices were deployed in applicable defects. Linear regression evaluated relationships between device size, defect size, and deployed dimensions. Six ASDOs of each size (27, 32, 37, 44, and 48 mm) were deployed in all applicable defects. There was significant ASDO size-defect size interaction in determining deployed ASDO diameter. Diameter was positively associated with defect size for 48-mm (B = 0.13, p < 0.001) and 44-mm (B = 0.11, p < 0.001) ASDOs, while no association was seen for 27-mm, 32-mm, or 37-mm ASDOs. No such interaction existed for deployed width or left-right disc difference. Controlling for ASDO size, width (B = - 0.12, p < 0.001) and left-right disc difference (B = - 0.06, p < 0.001) were negatively associated with defect size. In smaller defects, the 44-mm and 48-mm ASDOs display progressive diameter foreshortening, and all devices display progressive increase in width and left-right disc asymmetry. Anticipating the degree of diameter foreshortening may be critical when attempting closure of fenestrated lesions and/or in patients with limited total atrial septal length.

Giant Eustachian valve and Thebesian valve-A highly deceptive structures as atrial septal defect rims.

The Eustachian valve (EV) of the inferior vena cava and the Thebesian valve (TV) of the coronary sinus are incompletely regressed structures of embryonic sinus venosus. In the majority of cases, the EV and TV disappear completely after birth or are represented only by a thin crescentic fold. On echocardiography, these vestiges may mimic abnormal structures. We report a case with giant EV and TV which were initially misinterpreted as rims of an atrial septal defect (ASD) leading to the false diagnosis of ostium secundum ASD.

Differences in morbidity and mortality in Down syndrome are related to the type of congenital heart defect.

Morbidity and mortality in Down syndrome (DS) are mainly related to congenital heart defects (CHDs). While CHDs with high prevalence in DS (typical CHDs), such as endocardial cushion defects, have been extensively described, little is known about the impact of less common CHDs (atypical CHDs), such as aortic coarctation and univentricular hearts. In our single-center study, we analyzed, in observational, retrospective manner, data regarding cardiac features, surgical management, and outcomes of a cohort of DS patients. Literature review was performed to investigate previously reported studies on atypical CHDs in DS. Patients with CHDs were subclassified as having typical or atypical CHDs. Statistical analysis was performed for comparison between the groups. The study population encompassed 859 DS patients, 72.2% with CHDs, of which 4.7% were atypical. Statistical analysis showed a significant excess in multiple surgeries, all-cause mortality and cardiac mortality in patients with atypical CHDs (p = .0067, p = .0038, p = .0001, respectively). According to the Kaplan-Meier method, survival at 10 and 40 years was significantly higher in typical CHDs (99 and 98% vs. 91 and 84%, log rank <0.05). Among atypical CHDs, it seems that particularly multiple complex defects in univentricular physiology associate with a worse outcome. This may be due to the surgical difficulty in managing univentricular hearts with multiple defects concurring to the clinical picture or to the severity of associated defects themselves. Further studies need to address this specific issue, also considering the higher pulmonary pressures, infective complications, and potential comorbidities in DS patients.

Familial dilated cardiomyopathy associated with pathogenic TBX5 variants: Expanding the cardiac phenotype associated with Holt-Oram syndrome.

Holt-Oram syndrome (HOS) is a rare, autosomal dominant disorder caused by heterozygous pathogenic variants in cardiac T-box transcription factor, TBX5. Classically, it is associated with upper limb malformations and variable cardiac abnormalities. Limb manifestations are considered to be invariably present, ranging in severity from limitation in movement, to triphalangeal thumbs, absent thumbs, shortened forearms, or phocomelia. Cardiac involvement is characterized by congenital heart defects, most commonly septal structural malformations, and conduction system disease. Recently, novel TBX5 variants have also been reported in association with dilated cardiomyopathy (DCM). In this context, we report eight individuals from four unrelated families, in whom pathogenic variants in TBX5 segregated with an atypical HOS phenotype. Affected individuals exhibit relatively mild skeletal features of HOS, with a predominant cardiac phenotype, which includes several individuals affected by non-ischaemic DCM. To our knowledge, these represent the first reported cases of DCM in families with skeletal features of HOS, some of whom also harbored variants previously linked to a classical HOS phenotype (p. Arg279* and p.Arg237Gln). This finding supports diverse roles of TBX5 in cardiovascular development and function, and confirms the importance of long-term cardiac surveillance for individuals affected by HOS. Furthermore, these families highlight the wide phenotypic variability of HOS, which may include comparatively mild upper limb findings in respect to cardiac manifestations.

Long-Term Follow-Up of Transthoracic Echocardiography-Guided Transcatheter Closure of Large Atrial Septal Defects (≥ 30 mm) Using the SHSMA Occluder.

Transcatheter closure of large atrial septal defects (ASDs) remains controversial. The aim of this study was to evaluate the feasibility and safety of transthoracic echocardiography (TTE)-guided transcatheter closure of large ASDs. Patients with large secundum ASDs (≥ 30 mm) who underwent device closure were retrospectively reviewed. TTE was performed to guide ASD occluder positioning and assess the immediate and long-term outcomes. A total of 60 patients (median age 43.5 years, range 15-78 years) were enrolled in the study. The median ASD size was 35 mm (range 30-42 mm). Mild to moderate pulmonary hypertension was observed in 36 patients (60%). Thirty-one patients (51.7%) had one short rim, and 18 patients (30.0%) had two deficient rims. Placement of the device was successful in 57 patients (95%), and the median device size was 42 mm (range 40-50 mm). Dislodgement of the device occurred in three patients with two deficient rims: a larger device was redeployed in one case, and two patients required surgical repair. During a median follow-up of 37 months (range 6-83 months), no residual shunts, erosion, or embolization were noted, and pulmonary hypertension resolved in 75% of the patients. Thus t vast majority (95%) of large ASDs can be successfully closed percutaneously using the Chinese-made Shanghai Shape Memory Alloy (SHSMA) occluder under TTE guidance. Long-term follow-up showed that transcatheter closure could become a safe and effective alternative to surgery in select large ASDs.

Nr2f1a balances atrial chamber and atrioventricular canal size via BMP signaling-independent and -dependent mechanisms.

Determination of appropriate chamber size is critical for normal vertebrate heart development. Although Nr2f transcription factors promote atrial maintenance and differentiation, how they determine atrial size remains unclear. Here, we demonstrate that zebrafish Nr2f1a is expressed in differentiating atrial cardiomyocytes. Zebrafish nr2f1a mutants have smaller atria due to a specific reduction in atrial cardiomyocyte (AC) number, suggesting it has similar requirements to Nr2f2 in mammals. Furthermore, the smaller atria in nr2f1a mutants are derived from distinct mechanisms that perturb AC differentiation at the chamber poles. At the venous pole, Nr2f1a enhances the rate of AC differentiation. Nr2f1a also establishes the atrial-atrioventricular canal (AVC) border through promoting the differentiation of mature ACs. Without Nr2f1a, AVC markers are expanded into the atrium, resulting in enlarged endocardial cushions (ECs). Inhibition of Bmp signaling can restore EC development, but not AC number, suggesting that Nr2f1a concomitantly coordinates atrial and AVC size through both Bmp-dependent and independent mechanisms. These findings provide insight into conserved functions of Nr2f proteins and the etiology of atrioventricular septal defects (AVSDs) associated with NR2F2 mutations in humans.

KLF13 is a genetic modifier of the Holt-Oram syndrome gene TBX5.

TBX5, a member of the T-box family of transcription factors, is a dosage sensitive regulator of heart development. Mutations in TBX5 are responsible for Holt-Oram Syndrome, an autosomal dominant disease with variable and partially penetrant cardiac defects suggestive of the existence of genetic and environmental modifiers. KLF13, a member of the Krüppel-like family of zinc finger proteins is co-expressed with TBX5 in several cardiac cells including atrial cardiomyocytes and cells of the interatrial septum. We report that KLF13 interacts physically and functionally with TBX5 to synergistically activate transcription of cardiac genes. We show that TBX5 contacts KLF13 via its T-domain and find that several disease-causing mutations therein have decreased KLF13 interaction. Whereas Klf13 heterozygote mice have no detectable cardiac defects, loss of a Klf13 allele in Tbx5 heterozygote mice significantly increases the penetrance of TBX5-dependent cardiac abnormalities including atrial, atrial-ventricular and ventricular septal defects. The results reveal for the first time combinatorial interaction between a T-box protein and a KLF family member and its importance for heart and possibly other organ development. The data also suggest that, in human, KLF13 may be a genetic modifier of the Holt-Oram Syndrome gene TBX5.

Mosaic trisomy 22 in a 4-year-old boy with congenital heart disease and general hypotrophy: A case report.

BACKGROUND: Trisomy 22 mosaicism is a rare autosomal anomaly with survival compatibility. Recognition of the complete trisomy 22 which is incompatible with life from the mosaic form is critical for genetic counseling. Affected mosaic cases have prevalent clinical presentations such as webbed neck, developmental delay, abnormal ears, cardiac disorders, and microcephaly. Phenotype of these patients is milder than full chromosomal aneuploidy, and the severity of the phenotype depends on the count of trisomic cells. We describe a 4-year-old boy with mosaic trisomy 22 from healthy parents and no family history of any genetic disorders in the pedigree. METHOD AND RESULTS: The patient had determined dysmorphic clinical features including facial asymmetry, cleft palate, gastroenteritis, hydronephrosis, developmental delay, genital anomalies, dysplastic toenails, flattened nasal bridge, congenital heart defect, hearing loss, cryptorchidism, and hypotonic muscle. He is the first reported with hypothyroidism and larynx wall thickness in worldwide and the first with atrial septal defect (ASD) from Iran. Chromosomal analyses using G-banding indicated a de novo Mos 47,XY,+22(6)/46,XY(44) karyotype with no other chromosomal structural changes. CONCLUSIONS: Our observations confirm the importance of cytogenetic analyses for determining the cause of congenital anomalies and provide a useful genetic counseling. In addition, due to the fact that some of mosaic trisomy 22 features are unavoidable such as CHD and general hypotrophy, we suggest including echocardiography test for early diagnosis during the clinical assessment.

Tbx5 Buffers Inherent Left/Right Asymmetry Ensuring Symmetric Forelimb Formation.

The forelimbs and hindlimbs of vertebrates are bilaterally symmetric. The mechanisms that ensure symmetric limb formation are unknown but they can be disrupted in disease. In Holt-Oram Syndrome (HOS), caused by mutations in TBX5, affected individuals have left-biased upper/forelimb defects. We demonstrate a role for the transcription factor Tbx5 in ensuring the symmetric formation of the left and right forelimb. In our mouse model, bilateral hypomorphic levels of Tbx5 produces asymmetric forelimb defects that are consistently more severe in the left limb than the right, phenocopying the left-biased limb defects seen in HOS patients. In Tbx hypomorphic mutants maintained on an INV mutant background, with situs inversus, the laterality of defects is reversed. Our data demonstrate an early, inherent asymmetry in the left and right limb-forming regions and that threshold levels of Tbx5 are required to overcome this asymmetry to ensure symmetric forelimb formation.

Congenital heart diseases and their association with the variant distribution features on susceptibility genes.

Congenital heart disease (CHD), one of the causes of childhood morbidity and mortality, is mainly triggered by a combination of environmental and genetic factors. Several susceptible genes, such as NKX2-5, GATA4 and TBX5, have been reported as closely related to heart and vessel development. CHD subtypes are classified into diverse clinical phenotypes, such as atrial septal defects (ASD), ventricular septal defects (VSD), tetralogy of Fallot (TOF), and Holt-Oram syndrome (HOS). Here, we summarize the associations of the genetic variants in these three genes with CHD subtypes. CHD-associated variants of NKX2-5 locate mainly in the tinman domain and the homeodomain. Mutations in the homeodomain are correlated with ASD and atrioventricular (AV) block subtypes. VSD-associated variants of GATA4 are mainly at its terminal ends. Variants of TBX5 gene are primarily in exons 3, 4, 5 and 7 and highly associated with HOS subtype. Hence, the variant distribution of NKX2-5, GATA4 and TBX5 are tightly associated with particular CHD subtypes. Further structure-modelling analysis revealed that these mutated amino acid residuals maintain their DNA-binding ability and structural stability. Therefore structural features of these genes may be used to predict the high risk of CHD subtypes in infants.

[Comparative study between cardiac catheterization intervention therapy and transthoracic small incision surgery for closure of congenital atrial septal defect by domestic occluder with echocardiographic monitoring].

OBJECTIVE: To evaluate the safety of cardiac catheterization intervention therapy and transthoracic small incision surgery in the occlusion bydomestic occluder under echocardiography guiding in patients with atrial septal defect (ASD).
 Methods: A total of 1 080 patients with ASD in the occlusion by domestic occluder were analyzed retrospectively, and the interventional treatment were performed in 734 cases through cardiac catheterization intervention therapy and 346 cases through transthoracic small incision surgery. The patients undergone cardiac catheterization intervention therapy were guided under the digital substraction angiography (DSA) and were monitored by transthoracic echocardiography (TTE) in the whole interventional process, and the efficacy was evaluated with TTE. The occlusion of transthoracic small incision surgery was guided under the transesophageal echocardiography (TEE), which was used to monitor the position of occluder and evaluate the efficacy immediately.
 Results: Two kinds of intervention in the occlusion by domestic occluder had achieved satisfactory results in patients with ASD. There was no statistically difference in the longest size of ASD between the 2 intervention methods, while there were statistically differences in the ratio between ASD longest diameter and atrial septal length, and the size of the occlusion, and the disparity between the size of the occluder and ASD longest diameter (D value), respectively (all P<0.05). When the size of arithmetic mean of the ASD was <30 mm, the success rate of the 2 methods was both 100%. When the size of arithmetic mean of the ASD was ≥30 mm, the success rate was 100% in the transthoracic small incision surgery and 50% in the cardiac catheterization intervention therapy.
 Conclusion: Domestic occluder is safe. Compared with the imported one, its cost is lower. When the size of the defects is same, the occlusion is smaller in the transthoracic small incision surgery compared with that in the cardiac catheterization intervention therapy. When the size of arithmetic mean of the ASD is ≥30 mm, the success rate of the transthoracic small incision surgery is higher compared with the cardiac catheterization intervention therapy. When the cardiac catheterization intervention therapy fails, the transthoracic small incision surgery may be a better choice.

An analysis of cardiac defects and surgical interventions in 84 cases with full trisomy 18.

Trisomy 18 (Edwards syndrome) is the second most common autosomal trisomy after trisomy 21. Medical issues commonly include cardiac defects, such as ventricular septal defect (VSD) and atrial septal defect (ASD). If untreated, these conditions can contribute to the associated infant mortality. The objective of the study was review parent-reported information on 84 cases with full trisomy 18 focusing on prenatal and postnatal assessment and confirmation of cardiac defects and on subsequent treatment with cardiac surgery and post-surgery outcomes. At birth, 65 parent responses indicated the presence of VSD (77.4%), 38 ASD (45.2%), and 50 patent ductus arteriosus (PDA) (59.5%). The presence of multiple cardiac defects was also analyzed including 25 cases with VSD, ASD, and PDA at birth. The total reduced to 18 at survey completion. Twenty-four cases had one or more cardiac defects repaired for a total of 34 corrective surgeries. Age at surgery varied from 2 weeks to 41 months of age with most performed under 1 year of age. Twenty-one cases were still living at the time of survey completion (87.5%). From these date we provide recommendations and implications.

Inferior and right-sided juxtaposition of the left atrial appendage with an unexpected type of inter-atrial communication.

We have re-investigated an unusual cardiac specimen with juxtaposition of the atrial appendages. The original description dates to 1962, when the autopsy was performed at the Children's Memorial Hospital in Chicago, now Ann & Robert H. Lurie Children's Hospital of Chicago. The heart was subsequently stored in the Farouk S. Idriss Cardiac Registry at the same institution. The specimen shows usual atrial arrangement, but with the morphologically left appendage juxtaposed in a rightward manner, passing behind the heart rather than through the transverse sinus so as to reach its location inferior to the morphologically right appendage. The heart also demonstrated an inter-atrial communication between the cavities of the juxtaposed left appendage and the morphologically right atrium. We provide a detailed description of the morphology, and provide images of this lesion, which to the best of our knowledge has not previously been described.