Molecular Pathways and Animal Models of Arrhythmias.

Arrhythmias account for over 300,000 annual deaths in the United States, and approximately half of all deaths are associated with heart disease. Mechanisms underlying arrhythmia risk are complex; however, work in humans and animal models over the past 25 years has identified a host of molecular pathways linked with both arrhythmia substrates and triggers. This chapter will focus on select arrhythmia pathways solved by linking human clinical and genetic data with animal models.

Autistic traits in myotonic dystrophy type 1 due to MBNL inhibition and RNA mis-splicing.

Tandem repeat expansions are enriched in autism spectrum disorder, including CTG expansion in the DMPK gene that underlines myotonic muscular dystrophy type 1. Although the clinical connection of autism to myotonic dystrophy is corroborated, the molecular links remained unknown. Here, we show a mechanistic path of autism via repeat expansion in myotonic dystrophy. We found that inhibition of muscleblind-like (MBNL) splicing factors by expanded CUG RNAs alerts the splicing of autism-risk genes during brain development especially a class of autism-relevant microexons. To provide in vivo evidence that the CTG expansion and MBNL inhibition axis leads to the presentation of autistic traits, we demonstrate that CTG expansion and MBNL-null mouse models recapitulate autism-relevant mis-splicing profiles and demonstrate social deficits. Our findings indicate that DMPK CTG expansion-associated autism arises from developmental mis-splicing. Understanding this pathomechanistic connection provides an opportunity for greater in-depth investigations of mechanistic threads in autism.

Study of pathogenic genes in a pedigree with familial dilated cardiomyopathy.

BACKGROUND: Dilated cardiomyopathy (DCM) is a genetically heterogeneous cardiac disorder characterized by left ventricular dilation and contractile dysfunction. The substantial genetic heterogeneity evident in patients with DCM contributes to variable disease severity and complicates overall prognosis, which can be very poor. AIM: To identify pathogenic genes in DCM through pedigree analysis. METHODS: Our research team identified a patient with DCM in the clinic. Through investigation, we found that the family of this patient has a typical DCM pedigree. High-throughput sequencing technology, next-generation sequencing, was used to sequence the whole exomes of seven samples in the pedigree. RESULTS: A novel and potentially pathogenic gene mutation-ANK2p.F3067L-was discovered. The mutation was completely consistent with the clinical information for this DCM pedigree. Sanger sequencing was used to further verify the locus of the mutation in pedigree samples. These results were consistent with those of high-throughput sequencing. CONCLUSIONS: ANK2p.F3067L is considered a novel and potentially pathogenic gene mutation in DCM.

ANK2 as a novel predictive biomarker for immune checkpoint inhibitors and its correlation with antitumor immunity in lung adenocarcinoma.

BACKGROUND: Immune checkpoint inhibitors (ICIs) have been shown to significantly improve the survival of patients with advanced lung adenocarcinoma (LUAD). However, only limited proportion of patients could benefit from ICIs. Novel biomarkers with strong predictability are needed for clinicians to maximize the efficacy of ICIs. Our study aimed to identify potential biomarkers predicting ICIs efficacy in LUAD. METHODS: The Cancer Genome Atlas (TCGA) PanCancer Atlas studies in cBioportal were used to evaluate the mutation frequency of ANK2 across multiple cancers. Clinical and mutational data for LUAD from ICIs-treated cohorts (Hellmann et al. and Rizvi et al.) were collected to explore the correlation between ANK2 mutation and clinical outcomes. In addition, the relationship between ANK2 expression and clinical outcomes was analyzed using LUAD data from TCGA and Gene Expression Omnibus. Furthermore, the impact of ANK2 mutation and expression on the tumor immune microenvironment of LUAD was analyzed using TCGA and TISIDB databases. RESULTS: Patients with ANK2 mutation benefited more from ICIs. In ICIs-treated cohort, prolonged progression-free survival (PFS) (median PFS: NR (not reached) vs. 5.42 months, HR (hazard ratio) 0.31, 95% CI 0.18-0.54; P = 0.0037), improved complete response rate (17.65% vs. 1.85%, P = 0.0402), and improved objective response rate (64.71% vs. 24.07%, P = 0.0033) were observed in LUAD patients with ANK2 mutation compared to their wild-type counterparts. Regarding ANK2 expression, it was observed that ANK2 expression was decreased in LUAD (P < 0.05) and a higher level of ANK2 expression was associated with longer overall survival (HR 0.69, 95% CI 0.52-0.92; P = 0.012) in TCGA LUAD cohort. Moreover, ANK2 mutation or higher ANK2 expression correlated with enhanced antitumor immunity and "hot" tumor microenvironment in LUAD, which could be potential mechanisms that ANK2 mutation facilitated ICIs therapy and patients with higher ANK2 expression survived longer. CONCLUSION: Our findings suggest that ANK2 mutation or increased ANK2 expression may serve as a favorable biomarker for the efficacy of ICIs in patients with LUAD.

Taquicardia ventricular asociada a mutación en el gen de ankirina B / Ventricular tachycardia associated to a mutation in the ankyrin B gen

Resumen Se presenta el caso de una mujer de 14 años, con taquicardiomiopatía secundaria a taquicardia ventricular. Se evidenció la presencia de una variante de significado incierto en el gen ANK2, por lo que se consideró un posible síndrome de ankirina B. La paciente fue tratada con éxito a través de ablación con radiofrecuencia. Tras dicho procedimiento, tuvo recuperación completa de su función ventricular izquierda y resolución de los complejos ventriculares prematuros y los episodios de taquicardia ventricular.

Abstract We report a case of a 14-year-old with tachycardiomyopathy due to ventricular tachycardia. A variant of uncertain significance of the ANK2 gene was identified, which is suggestive of a possible ankyrin-B syndrome. The patient underwent a successful radiofrequency ablation. After the procedure, the patient completely recovered her left ventricular function and there was resolution of the premature ventricular complexes and ventricular tachycardia.

Theory and Applications of the (Cardio) Genomic Fabric Approach to Post-Ischemic and Hypoxia-Induced Heart Failure.

The genomic fabric paradigm (GFP) characterizes the transcriptome topology by the transcripts' abundances, the variability of the expression profile, and the inter-coordination of gene expressions in each pathophysiological condition. The expression variability analysis provides an indirect estimate of the cell capability to limit the stochastic fluctuations of the expression levels of key genes, while the expression coordination analysis determines the gene networks in functional pathways. This report illustrates the theoretical bases and the mathematical framework of the GFP with applications to our microarray data from mouse models of post ischemic, and constant and intermittent hypoxia-induced heart failures. GFP analyses revealed the myocardium priorities in keeping the expression of key genes within narrow intervals, determined the statistically significant gene interlinkages, and identified the gene master regulators in the mouse heart left ventricle under normal and ischemic conditions. We quantified the expression regulation, alteration of the expression control, and remodeling of the gene networks caused by the oxygen deprivation and determined the efficacy of the bone marrow mono-nuclear stem cell injections to restore the normal transcriptome. Through the comprehensive assessment of the transcriptome, GFP would pave the way towards the development of personalized gene therapy of cardiac diseases.

Autism-associated ANK2 regulates embryonic neurodevelopment.

Autism spectrum disorder (ASD) is a neurodevelopmental condition characterized by altered social communication, restricted interests, and stereotypic behaviors. Although the molecular and cellular pathogeneses of ASD remain elusive, impaired neural stem cell differentiation and neuronal migration during cortical development are suggested to be critically involved in ASD. ANK2, which encodes for a cytoskeletal scaffolding protein involved in recruiting membrane proteins into specialized membrane domains, has been identified as a high-confidence ASD risk gene. However, the role of ANK2 in early neural development remains unclear. In this study, we analyzed the role of ANK2 in the cerebral cortex of developing mouse using in utero electroporation. We provide evidence suggesting that ANK2 regulates neural stem cell differentiation and neuronal migration in the embryonic cerebral cortex, where Ank2 is highly expressed. We also demonstrated that Ank2 knockdown alters the expression of genes involved in neural development. Taken together, these results support the view that ANK2 haploinsufficiency in patients may impair neural development, resulting in an increased risk of ASD. Our study findings provide new insights into the molecular and cellular pathogenesis of ASD, given that among high-confidence ASD genes, ANK2 is rare in that it encodes for a scaffolding protein for the membrane protein complex required for neuronal functions.

Giant ankyrin-B mediates transduction of axon guidance and collateral branch pruning factor sema 3A.

Variants in the high confident autism spectrum disorder (ASD) gene ANK2 target both ubiquitously expressed 220 kDa ankyrin-B and neurospecific 440 kDa ankyrin-B (AnkB440) isoforms. Previous work showed that knock-in mice expressing an ASD-linked Ank2 variant yielding a truncated AnkB440 product exhibit ectopic brain connectivity and behavioral abnormalities. Expression of this variant or loss of AnkB440 caused axonal hyperbranching in vitro, which implicated AnkB440 microtubule bundling activity in suppressing collateral branch formation. Leveraging multiple mouse models, cellular assays, and live microscopy, we show that AnkB440 also modulates axon collateral branching stochastically by reducing the number of F-actin-rich branch initiation points. Additionally, we show that AnkB440 enables growth cone (GC) collapse in response to chemorepellent factor semaphorin 3 A (Sema 3 A) by stabilizing its receptor complex L1 cell adhesion molecule/neuropilin-1. ASD-linked ANK2 variants failed to rescue Sema 3A-induced GC collapse. We propose that impaired response to repellent cues due to AnkB440 deficits leads to axonal targeting and branch pruning defects and may contribute to the pathogenicity of ANK2 variants.

Structural Basis Underlying Strong Interactions between Ankyrins and Spectrins.

Ankyrins (encoded by ANK1/2/3 corresponding to Ankyrin-R/B/G or AnkR/B/G), via binding to spectrins, connect plasma membranes with actin cytoskeleton to maintain mechanical strengths and to modulate excitabilities of diverse cells such as neurons, muscle cells, and erythrocytes. Cellular and genetic evidences suggest that each isoform of ankyrins pairs with a specific ß-spectrin in discrete subcellular membrane microdomains for distinct functions, although the molecular mechanisms underlying such ankyrin/ß-spectrin pairings are unknown. In this study, we discover that a conserved and short extension N-terminal to the ZU5N-ZU5C-UPA tandem (exZZU) is critical for each ankyrin to bind to ß-spectrins with high affinities. Structures of AnkB/G exZZU in complex with spectrin repeats13-15 of ß2/ß4-spectrins solved here reveal that the extension sequence of exZZU forms an additional ß-strand contributing to the structural stability and enhanced affinity of each ZU5N/spectrin repeat interaction. The complex structures further reveal that the UPA domain of exZZU directly participates in spectrin binding. Formation of the exZZU supramodule juxtaposes the ZU5N and UPA domains for simultaneous interacting with spectrin repeats 14 and 15. However, our biochemical and structural investigations indicate that the direct and strong interactions between ankyrins and ß-spectrins do not appear to determine their pairing specificities. Therefore, there likely exists additional mechanism(s) for modulating functional pairings between ankyrins and ß-spectrins in cells.

Wolff-Parkinson-White syndrome: De novo variants and evidence for mutational burden in genes associated with atrial fibrillation.

BACKGROUND: Wolff-Parkinson-White (WPW) syndrome is a relatively common arrhythmia affecting ~1-3/1,000 individuals. Mutations in PRKAG2 have been described in rare patients in association with cardiomyopathy. However, the genetic basis of WPW in individuals with a structurally normal heart remains poorly understood. Sudden death due to atrial fibrillation (AF) can also occur in these individuals. Several studies have indicated that despite ablation of an accessory pathway, the risk of AF remains high in patients compared to general population. METHODS: We applied exome sequencing in 305 subjects, including 65 trios, 80 singletons, and 6 multiple affected families. We used de novo analysis, candidate gene approach, and burden testing to explore the genetic contributions to WPW. RESULTS: A heterozygous deleterious variant in PRKAG2 was identified in one subject, accounting for 0.6% (1/151) of the genetic basis of WPW in this study. Another individual with WPW and left ventricular hypertrophy carried a known pathogenic variant in MYH7. We found rare de novo variants in genes associated with arrhythmia and cardiomyopathy (ANK2, NEBL, PITX2, and PRDM16) in this cohort. There was an increased burden of rare deleterious variants (MAF ≤ 0.005) with CADD score ≥ 25 in genes linked to AF in cases compared to controls (P = .0023). CONCLUSIONS: Our findings show an increased burden of rare deleterious variants in genes linked to AF in WPW syndrome, suggesting that genetic factors that determine the development of accessory pathways may be linked to an increased susceptibility of atrial muscle to AF in a subset of patients.

Mechanisms and Alterations of Cardiac Ion Channels Leading to Disease: Role of Ankyrin-B in Cardiac Function.

Ankyrin-B (encoded by ANK2), originally identified as a key cytoskeletal-associated protein in the brain, is highly expressed in the heart and plays critical roles in cardiac physiology and cell biology. In the heart, ankyrin-B plays key roles in the targeting and localization of key ion channels and transporters, structural proteins, and signaling molecules. The role of ankyrin-B in normal cardiac function is illustrated in animal models lacking ankyrin-B expression, which display significant electrical and structural phenotypes and life-threatening arrhythmias. Further, ankyrin-B dysfunction has been associated with cardiac phenotypes in humans (now referred to as "ankyrin-B syndrome") including sinus node dysfunction, heart rate variability, atrial fibrillation, conduction block, arrhythmogenic cardiomyopathy, structural remodeling, and sudden cardiac death. Here, we review the diverse roles of ankyrin-B in the vertebrate heart with a significant focus on ankyrin-B-linked cell- and molecular-pathways and disease.

Downregulation of miR-146a Contributes to Cardiac Dysfunction Induced by the Tyrosine Kinase Inhibitor Sunitinib.

The main adverse effect of tyrosine kinase inhibitors, such as sunitinib, is cardiac contractile dysfunction; however, the molecular mechanisms of this effect remain largely obscure. MicroRNAs (miRNAs) are key regulatory factors in both cardiovascular diseases and the tyrosine kinase pathway. Therefore, we analyzed the differential expression of miRNAs in the myocardium in mice after exposure to sunitinib using miRNA microarray. A significant downregulation of miR-146a was observed in the myocardium of sunitinib-treated mice, along with a 20% decrease in left ventricle ejection fraction (LVEF). The downregulation of miR-146a was further validated by RT-qPCR. Among the potential targets of miR-146a, we focused on Pln and Ank2, which are closely related to cardiac contractile dysfunction. Results of luciferase reporter assay confirmed that miR-146a directly targeted the 3' untranslated region of Pln and Ank2. Significant upregulation of PLN and ANK2 at the mRNA and protein levels was observed in the myocardium of sunitinib-treated mice. Cardiac-specific overexpression of miR-146a prevented the deteriorate effect of SNT on calcium transients, thereby alleviating the decreased contractility of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). SiRNA knockdown of PLN or ANK2 prevented sunitinib-induced suppression of contractility in hiPSC-CMs. Therefore, our in vivo and in vitro results showed that sunitinib downregulated miR-146a, which contributes to cardiac contractile dysfunction by regulating the downstream targets PLN and ANK2, and that upregulation of miR-146a alleviated the inhibitory effect of SNT on cardiac contractility. Thus, miR-146a could be a useful protective agent against sunitinib-induced cardiac dysfunction.

ANK2 autism mutation targeting giant ankyrin-B promotes axon branching and ectopic connectivity.

Giant ankyrin-B (ankB) is a neurospecific alternatively spliced variant of ANK2, a high-confidence autism spectrum disorder (ASD) gene. We report that a mouse model for human ASD mutation of giant ankB exhibits increased axonal branching in cultured neurons with ectopic CNS axon connectivity, as well as with a transient increase in excitatory synapses during postnatal development. We elucidate a mechanism normally limiting axon branching, whereby giant ankB localizes to periodic axonal plasma membrane domains through L1 cell-adhesion molecule protein, where it couples microtubules to the plasma membrane and prevents microtubule entry into nascent axon branches. Giant ankB mutation or deficiency results in a dominantly inherited impairment in selected communicative and social behaviors combined with superior executive function. Thus, gain of axon branching due to giant ankB-deficiency/mutation is a candidate cellular mechanism to explain aberrant structural connectivity and penetrant behavioral consequences in mice as well as humans bearing ASD-related ANK2 mutations.

ANK2 functionally interacts with KCNH2 aggravating long QT syndrome in a double mutation carrier.

Pathogenic long QT mutations often comprise high phenotypic variability and particularly variants in ANK2 (long QT syndrome 4) frequently lack QT prolongation. We sought to elucidate the genetic and functional background underlying the clinical diversity in a 3-generation family with different cardiac arrhythmias. Next-generation sequencing-based screening of patients with QT prolongation identified the index patient of the family carrying an ANK2-E1813K variant and a previously uncharacterized KCNH2-H562R mutation in a double heterozygous conformation. The patient presented with a severe clinical phenotype including a markedly prolonged QTc interval (544 ms), recurrent syncope due to Torsade de Pointes tachycardias, survived cardiopulmonary resuscitation, progressive cardiac conduction defect, and atrial fibrillation. Evaluation of other family members identified a sister and a niece solely carrying the ANK2-E1813K variant, who showed age-related conduction disease. An asymptomatic second sister solely carried the KCNH2-H562R mutation. Voltage-clamp recordings in Xenopus oocytes revealed that KCNH2-H562R subunits were non-functional but did not exert dominant-negative effects on wild-type subunits. Expression of KCNH2-H562R in HEK293 cells showed a trafficking deficiency. Co-expression of the C-terminal regulatory domain of ANK2 in Xenopus oocytes revealed that ANK2-E1813K diminished currents mediated by the combination of wild-type and H562R KCNH2 subunits. Our data suggest that ANK2 functionally interacts with KCNH2 leading to a stronger current suppression and marked aggravation of long QT syndrome in the patient carrying variants in both proteins.

All Along the Watchtower: a Case of Long QT Syndrome Misdiagnosis Secondary to Genetic Testing Misinterpretation.

Clinical genetics services continue to expand into diverse medical specialties. An ever-increasing number of non-genetics providers are independently ordering genetic tests, interpreting results, and at times, making diagnoses leading to patient care recommendations. Non-genetics healthcare providers can help increase patient access to these services, but a potential pitfall occurs when these providers either do not have adequate expertise with genetic variant interpretation or do not have access to multi-disciplinary teams including genetic counselors or clinical geneticists for advanced review. In the cardiology setting, variant misinterpretation can lead to misattribution of disease risk, unnecessary treatments or management, and potentially adverse psychosocial and financial effects. To address this, case reports and series are needed to highlight variant misinterpretation and misdiagnoses, including discussion of possible solutions and best practices for avoidance. This report details a child previously diagnosed with long QT syndrome type 4 by chromosomal microarray who was then subsequently managed for this disease by cardiac providers with insufficient expertise to critically review and question the genetic testing results. The patient was eventually referred to a pediatric electrophysiology team as part of a larger multidisciplinary cardiovascular genetics program, composed of specialist genetic counselors, cardiologists, and clinical geneticists. Advanced review and clinical evaluation raised concern about the initial genetic testing result and diagnosis. Complementary testing with a different modality to confirm or disconfirm the chromosome microarray result was performed, providing evidence that the original result reflected analytic error in the laboratory as well as interpretive error by the clinical geneticist and that the patient was misdiagnosed, and treated over the course of years, for long QT syndrome. This case shows the value of multidisciplinary teams caring for patients with inherited cardiovascular diseases.

The evolving role of ankyrin-B in cardiovascular disease.

Over the past decade, ankyrin-B has been identified as a prominent player in cardiac physiology. Ankyrin-B has a multitude of functions, with roles in expression, localization, and regulation of proteins critical for cardiac excitability, cytoskeletal integrity, and signaling. Furthermore, human ANK2 variants that result in ankyrin-B loss of function are associated with "ankyrin-B syndrome," a complex cardiac phenotype that may include bradycardia and heart rate variability, conduction block, atrial fibrillation, QT interval prolongation, and potentially fatal catecholaminergic polymorphic ventricular tachycardia. However, our understanding of the molecular mechanisms underlying ankyrin-B function at baseline and in disease is still not fully developed owing to the complexity of ankyrin-B gene regulation, number of ankyrin-B-associated molecules, multiple roles of ankyrin-B in the heart and other organs that modulate cardiac function, and a host of unexpected clinical phenotypes. In this review, we summarize known roles of ankyrin-B in the heart and the impact of ankyrin-B dysfunction in animal models and in human disease as well as highlight important new findings illustrating the complexity of ankyrin-B signaling.

A Novel Mechanism for Human Cardiac Ankyrin-B Syndrome due to Reciprocal Chromosomal Translocation.

BACKGROUND: Cardiac rhythm abnormalities are a leading cause of morbidity and mortality in developed countries. Loss-of-function variants in the ANK2 gene can cause a variety of cardiac rhythm abnormalities including sinus node dysfunction, atrial fibrillation and ventricular arrhythmias (called the "ankyrin-B syndrome"). ANK2 encodes ankyrin-B, a molecule critical for the membrane targeting of key cardiac ion channels, transporters, and signalling proteins. METHODS AND RESULTS: Here, we describe a family with a reciprocal chromosomal translocation between chromosomes 4q25 and 9q26 that transects the ANK2 gene on chromosome 4 resulting in loss-of-function of ankyrin-B. Select family members with ankyrin-B haploinsufficiency due to the translocation displayed clinical features of ankyrin-B syndrome. Furthermore, evaluation of primary lymphoblasts from a carrier of the translocation showed altered levels of ankyrin-B as well as a reduced expression of downstream ankyrin-binding partners. CONCLUSIONS: Thus, our data conclude that, similar to previously described ANK2 loss-of-function "point mutations", large chromosomal translocations resulting in ANK2 haploinsufficiency are sufficient to cause the human cardiac ankyrin-B syndrome. The unexpected ascertainment of ANK2 dysfunction via the discovery of a chromosomal translocation in this family, the determination of the familial phenotype, as well as the complexities in formulating screening and treatment strategies are discussed.

Hox Function Is Required for the Development and Maintenance of the Drosophila Feeding Motor Unit.

Feeding is an evolutionarily conserved and integral behavior that depends on the rhythmic activity of feeding muscles stimulated by specific motoneurons. However, critical molecular determinants underlying the development of the neuromuscular feeding unit are largely unknown. Here, we identify the Hox transcription factor Deformed (Dfd) as essential for feeding unit formation, from initial specification to the establishment of active synapses, by controlling stage-specific sets of target genes. Importantly, we found Dfd to control the expression of functional components of synapses, such as Ankyrin2-XL, a protein known to be critical for synaptic stability and connectivity. Furthermore, we uncovered Dfd as a potential regulator of synaptic specificity, as it represses expression of the synaptic cell adhesion molecule Connectin (Con). These results demonstrate that Dfd is critical for the establishment and maintenance of the neuromuscular unit required for feeding behavior, which might be shared by other group 4 Hox genes.

Structural basis of diverse membrane target recognitions by ankyrins.

Ankyrin adaptors together with their spectrin partners coordinate diverse ion channels and cell adhesion molecules within plasma membrane domains and thereby promote physiological activities including fast signaling in the heart and nervous system. Ankyrins specifically bind to numerous membrane targets through their 24 ankyrin repeats (ANK repeats), although the mechanism for the facile and independent evolution of these interactions has not been resolved. Here we report the structures of ANK repeats in complex with an inhibitory segment from the C-terminal regulatory domain and with a sodium channel Nav1.2 peptide, respectively, showing that the extended, extremely conserved inner groove spanning the entire ANK repeat solenoid contains multiple target binding sites capable of accommodating target proteins with very diverse sequences via combinatorial usage of these sites. These structures establish a framework for understanding the evolution of ankyrins' membrane targets, with implications for other proteins containing extended ANK repeat domains.