Association study of FLT4 and HYDIN single nucleotide polymorphisms with atrial septal defect susceptibility in the Han Chinese population of Southwest China.

BACKGROUND: Atrial septal defect (ASD) is a common form of congenital heart disease. Although several genes related to ASD have been found, the genetic factors of ASD remain unclear. This study aimed to evaluate the correlation between 10 candidate single nucleotide polymorphisms (SNPs) and sporadic atrial septal defects. METHODS: Based on the results of 34 individual whole exome sequences, 10 candidate SNPs were selected. In total, 489 ASD samples and 420 normal samples were collected. The 10 SNPs in the case group and the control group were identified through Snapshot genotyping technology. The χ2-test and unconditional regression model were used to evaluate the relationship between ASD and each candidate SNP. Haploview software was used to perform linkage disequilibrium and haplotype analysis. RESULTS: The χ2 results showed that the FLT4 rs383985 (P = 0.003, OR = 1.115-1.773), HYDIN rs7198975 (P = 0.04621, OR = 1.003-1.461), and HYDIN rs1774266 (P = 0.04621, OR = 1.003-1.461) alleles were significantly different between the control group and the case group (P < 0.05). Only the association with the FLT4 polymorphism was statistically significant after adjustment for multiple comparisons. CONCLUSION: These findings suggest that a possible molecular pathogenesis associated with sporadic ASD is worth exploring in future studies.

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.

TBX5 variants and cardiac phenotype: A systematic review of the literature and a novel variant.

T-Box Transcription Factor 5 (TBX5) variants are associated with Holt-Oram syndrome. Holt-Oram syndrome display phenotypic variability, regarding upper limb defects, congenital heart defects, and arrhythmias. To investigate the genotype-phenotype relationship between TBX5 variants and cardiac disease, we performed a systematic review of the literature. Through the systematic review we identified 108 variants in TBX5 associated with a cardiac phenotype in 277 patients. Arrhythmias were more frequent in patients with a missense variant (48% vs 30%, p = 0.009) and upper limb abnormalities were more frequent in patients with protein-truncating variants (85% vs 64%, p = 0.0008). We found clustering of missense variants in the T-box domain. Furthermore, we present a family with atrial septal defects. By whole exome sequencing, we identified a novel missense variant p.Phe232Leu in TBX5. The cardiac phenotype included atrial septal defect, arrhythmias, heart failure, and dilated cardiomyopathy. Clinical examination revealed subtle upper limb abnormalities. Thus, the family corresponds to the diagnostic criteria of Holt-Oram syndrome. We provide an overview of cardiac phenotypes associated with TBX5 variants and show an increased risk of arrhythmias associated to missense variants compared to protein-truncating variants. We report a novel missense variant in TBX5 in a family with an atypical Holt-Oram syndrome phenotype.

Holt-Oram syndrome.

BACKGROUND: Holt-Oram syndrome (HOS) is a rare genetic illness, which concerns disturbances in the appearance of the upper limbs, congenital heart malformations, and cardiac conduction diseases. HOS usually requires the implantation of a pacemaker, because of cardiac conduction disturbances. CASE REPORT: We present the case of a patient with HOS qualified for pacemaker implantation due to overt bradycardia. To prevent the development of heart failure in the future, the His-bundle pacing technique was used. The implantation was successful. In the control, after one year, the man remains in good condition. The pacing was over 90%, and the left ventricular ejection fraction (LVEF) was stable (60%). CONCLUSIONS: So far, there are no reports on which methods of stimulation are required when it comes to patients with HOS. His-bundle pacing technique is a new type of physiological pacing, which can avoid heart failure.

Case Report: A novel EPAS1 mutation in a case of paraganglioma complicated with polycythemia and atrial septal defect.

Background: Paraganglioma is a rare neuroendocrine tumor and is highly associated with hereditary susceptibility genes, often occurring as part of a genetic syndrome. The genetic heterogeneity of paraganglioma poses challenges in diagnosis, counseling, and clinical management. Case summary: We present the case of a 60-year-old woman with hypertension, atrial septal defect, and polycythemia, who experienced paroxysmal palpitations, sweating, headache, abdominal pain, nausea, and vomiting. Her blood pressure was severely unstable. Blood laboratory tests revealed elevated catecholamine levels, contrast-enhanced CT of her whole abdomen showed a round retroperitoneal mass with soft tissue density, and somatostatin receptor imaging (68Ga PET-CT) indicated a retroperitoneal mass with abnormally increased expression of somatostatin receptor. It is interesting to note that whole exome sequencing (WES) analyses on both blood and tumor samples revealed a novel EPAS1 mutation, specifically the c.2501A > G; p.Tyr834Cys variant, which has never been reported. The patient was diagnosed with paraganglioma and underwent successful Da Vinci robot-assisted laparoscopic resection of the retroperitoneal tumor. During a 3-month follow-up period, her blood pressure stabilized, and her symptoms significantly improved. Conclusion: This case reveals that the EPSA1 mutation may be the primary driver of paraganglioma complicated by atrial septal defect and polycythemia. Additionally, the utilization of Da Vinci robot-assisted laparoscopic surgery contributed to a favorable prognosis for the patient.

A rare case of isolated right atrial enlargement and TBX5 mutation associated with Holt-Oram syndrome.

Holt-Oram syndrome or atriodigital dysplasia is commonly associated with cardiac malformations, most often with defects of the muscular septum. We describe the case of a fetus referred for fetal cardiology evaluation in the setting of right atrial enlargement without tricuspid valve abnormalities with small muscular VSDs, and without other significant cardiac lesions. On serial fetal echocardiograms, isolated right atrial enlargement was persistent as was relative fetal bradycardia without apparent AV block or other signs of abnormal conduction. Limb or other anatomic abnormalities were also not visualized on prenatal scans. A postnatal diagnosis of Holt-Oram Syndrome was made. In the setting of isolated right atrial enlargement, we suggest a comprehensive sonographic search for upper limb abnormalities as well as genetic evaluation.

Cytogenetic study of subtypes of Down syndrome and its relation with pattern of congenital cardiac defects.

OBJECTIVE: To determine the frequency of subtypes of Down syndrome by karyotyping, and to establish the frequency of congenital cardiac defects in this population. METHODS: The cross-sectional study was conducted at the Department of Genetics, Children Hospital, Lahore, Pakistan, from June 2016 to June 2017, and comprised of Down Syndrome patients aged <15 years. They were subjected to karyotypic analysis for determining the subtype of the syndrome, and echocardiography of all cases was done for the assessment of congenital cardiac defects. The two findings was subsequently used to establish a relation between the subtypes and congenital cardiac defects. Data collected, entered and analyzed by the SPSS version 20.0. RESULTS: Among the 160 cases, trisomy 21 was found in 154(96.2%), translocation 5(3.1%) and mosaicism 1(0.6%). Overall, 63(39.4%) children had cardiac defects. Among such patients, patent ductus arteriosus was most common 25(39.7%), followed by ventricular septal defects24(38.1%), atrial septal defects16(25.4%), complete atrioventricular septal defects 8(12.7%), and Tetralogy of Fallot3(4.8%), while 6(9.5%) children had other defects. Atrial septal defects was the most common double defect 9(56.2%) and had the highest coexistence with patent ductus arteriosus in Down syndrome cases with congenital cardiac defects. CONCLUSIONS: In Trisomy 21, the most common cardiac defect was patent ductus arteriosus, followed by ventricular septal defects in isolated defects, whereas in mixed defects, atrial septal defects and patent ductus arteriosus were the highest.

NKX2-5 genetic mutation in a young woman with an atrial septal defect presenting with complete heart block: ICD or bradycardia pacemaker?

A woman in her 40s was admitted following syncope. The 12-lead ECG showed atrial fibrillation with slow ventricular response and suspected complete atrioventricular (AV) block. Cardiac monitoring demonstrated non-sustained monomorphic ventricular tachycardia (VT). Her medical history included surgical repair of an atrial septal defect (ASD) aged 4 years. The patient's mother died suddenly in her early 50s and also had an ASD. Given the patient's syncope, background of familial sudden cardiac death (SCD), suspicion of complete AV block and non-sustained VT, she received an implantable cardiac defibrillator (ICD). She underwent genetic testing, revealing a heterozygous NKX2-5 genetic mutation. The signature phenotype in NKX2-5 mutations is ASD with AV conduction disturbance and an increased risk of SCD secondary to ventricular arrhythmias or severe bradycardia. SCD has been described in NKX2-5 mutation carriers despite functioning permanent pacemakers (PPMs). Therefore, we propose implantation of a preventive ICD, as opposed to a PPM.

Clinical diversity and molecular mechanism of VPS35L-associated Ritscher-Schinzel syndrome.

PURPOSE: The Retriever subunit VPS35L is the third responsible gene for Ritscher-Schinzel syndrome (RSS) after WASHC5 and CCDC22. To date, only one pair of siblings have been reported and their condition was significantly more severe than typical RSS. This study aimed to understand the clinical spectrum and underlying molecular mechanism in VPS35L-associated RSS. METHODS: We report three new patients with biallelic VPS35L variants. Biochemical and cellular analyses were performed to elucidate disease aetiology. RESULTS: In addition to typical features of RSS, we confirmed hypercholesterolaemia, hypogammaglobulinaemia and intestinal lymphangiectasia as novel complications of VPS35L-associated RSS. The latter two complications as well as proteinuria have not been reported in patients with CCDC22 and WASHC5 variants. One patient showed a severe phenotype and the other two were milder. Cells established from patients with the milder phenotypes showed relatively higher VPS35L protein expression. Cellular analysis found VPS35L ablation decreased the cell surface level of lipoprotein receptor-related protein 1 and low-density lipoprotein receptor, resulting in reduced low-density lipoprotein cellular uptake. CONCLUSION: VPS35L-associated RSS is a distinct clinical entity with diverse phenotype and severity, with a possible molecular mechanism of hypercholesterolaemia. These findings provide new insight into the essential and distinctive role of Retriever in human development.

The clinical spectrum of a nonsense mutation in KAT6A: a case report.

KAT6A syndrome is an autosomal dominant genetic disorder associated with intellectual disability due to mutations in the lysine acetyltransferase 6A (KAT6A) gene. There are some differences in phenotype between KAT6A gene variants. This current case report describes a 1-month-old male infant that had a nonsense mutation in the KAT6A gene. Neither of his parents had the mutation. The proband had feeding difficulties and a physical examination revealed the following: moderate dysphagia, hypoplastic laryngeal cartilage, poor audio-visual response, poor head-up ability, no active grasping awareness, microcephaly, high arched palate and he was significantly behind other children of the same age. Echocardiography showed that the foramen ovale was not closed. He was diagnosed with atrial septal defect (ASD) when 2 years old. The patient received ASD repair at 32 months of age. Head colour Doppler ultrasonography and brain magnetic resonance imaging showed cysts in the right ventricle and choroid plexus, which returned to normal at 2 years of age. This current case demonstrates that immediate surgery should be considered in newborns with KAT6A syndrome presenting with a heart malformation. A new KAT6A syndrome phenotype is described in this current case report, which requires early diagnosis and treatment.

Identification and functional analysis of genetic variants of ISL1 gene promoter in human atrial septal defects.

BACKGROUND: Atrial septal defect (ASD) is a common type of congenital heart disease. A gene promoter plays pivotal role in the disease development. This study was designed to investigate the pathological role of variants of the ISL1 gene promoter region in ASD patients. METHODS: Total DNA extracted from 625 subjects, including 332 ASD patients and 293 healthy controls, was sequenced to identify variants in the promoter region of ISL1 gene. Further functional analyses of the variants were performed with dual luciferase reporter assay and electrophoretic mobility shift assay (EMSA). All possible binding sites of transcription factor affected by the identified variants were predicted using the JASPAR database. RESULTS: Four variants in the ISL1 gene promoter were found only in patients with ASD by sequencing. Three of the four variants [g.4923 G > C (rs541081886), g.5079 A > G (rs1371835943) and g.5309 G > A (rs116222082)] significantly decreased the transcriptional activities compared with the wild-type ISL1 gene promoter (p < 0.05). The EMSA revealed that these variants [g.4923 G > C (rs541081886), g.5079 A > G (rs1371835943) and g.5309 G > A (rs116222082)] in the ISL1 gene promoter affected the number and affinity of binding sites of transcription factors. Further analysis with the online JASPAR database demonstrated that a cluster of putative binding sites for transcription factors may be altered by these variants. CONCLUSIONS: These sequence variants identified from the promoter region of ISL1 gene in ASD patients are probably involved in the development of ASD by affecting the transcriptional activity and altering ISL1 levels. Therefore, these findings may provide new insights into the molecular etiology and potential therapeutic strategy of ASD.

Functional analysis of HECA variants identified in congenital heart disease in the Chinese population.

BACKGROUND: Congenital heart disease (CHD) is a class of cardiovascular defects that includes septal defects, outflow tract abnormalities, and valve defects. Human homolog of Drosophila headcase (HECA) is a novel cell cycle regulator whose role in CHD has not been elucidated. This is the first study to determine the frequency of HECA mutations in patients with CHD and the association between HECA variants and CHD. METHODS: In this study, we identified a candidate gene, HECA, by whole-exome sequencing of an atrial septal defect family. To investigate the association between HECA variants and CHD risk, targeted exon sequencing was conducted in 689 individuals with sporadic CHD. We further analyzed the effect of HECA gene abnormalities on cardiomyocyte phenotype behavior and related signaling pathways by Western blotting, reverse transcription-quantitative polymerase chain reaction, and scratch assay. RESULTS: We found a novel de novo mutation, c.409_410insA (p. W137fs), in the HECA gene and identified five rare deleterious variants that met the filtering criteria in 689 individuals with sporadic CHD. Fisher's exact test revealed a significant association between HECA variations and CHD compared with those in gnomADv2-East Asians(p = 0.0027). Further functional analysis suggested that the variant p. W137fs resulted in a deficiency of the normal HECA protein, and HECA deficiency altered AC16 cell cycle progression, increased cell proliferation, and migration, and promoted the activation of the PDGF-BB/PDGFRB/AKT pathway. CONCLUSIONS: Our study identified HECA and its six rare variants, expanding the spectrum of genes associated with CHD pathogenesis in the Chinese population.

High-risk genes involved in common septal defects of congenital heart disease.

The septation defect is one of the main categories of congenital heart disease (CHD). They can affect the septation of the atria leading to atrial septal defect (ASD), septation of ventricles leading to ventricular septal defect (VSD), and formation of the central part of the heart leading to atrioventricular septal defect (AVSD). Disruption of critical genetic factors involved in the proper development of the heart structure leads to CHD manifestation. Because of this, to identify the high-risk genes involved in common septal defects, a comprehensive search of the literature with the help of databases and the WebGestalt analysis tool was performed. The high-risk genes identified in the analysis were checked in 16 Indian whole-exome sequenced samples, including 13 VSD and three Tetralogy of Fallot for in silico validation. This data revealed three variations in GATA4, i.e., c.C1223A at exon 6: c.C602A and c.C1220A at exon 7; and one variation in MYH6, i.e., c.G3883C at exon 28 in two VSD cases. This study supports previously published studies that suggested GATA4 and MYH6 as the high-risk genes responsible for septal defects. Thus, this study contributes to a better understanding of the genes involved in heart development by identifying the high-risk genes and interacting proteins in the pathway.

A Novel Splicing Mutation c.335-1 G > A in the Cardiac Transcription Factor NKX2-5 Leads to Familial Atrial Septal Defect Through miR-19 and PYK2.

Mutations of NKX2-5 largely contribute to congenital heart diseases (CHDs), especially atrial septal defect (ASD). We identified a novel heterozygous splicing mutation c.335-1G > A in NKX2-5 gene in an ASD family via whole exome sequencing (WES) and linkage analysis. Utilizing the human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (hiPSC-CMs) as a disease model, we showed that haploinsufficiency of NKX2-5 contributed to aberrant orchestration of apoptosis and proliferation in ASD patient-derived hiPSC-CMs. RNA-seq profiling and dual-luciferase reporter assay revealed that NKX2-5 acts upstream of PYK2 via miR-19a and miR-19b (miR-19a/b) to regulate cardiomyocyte apoptosis. Meanwhile, miR-19a/b are also downstream mediators of NKX2-5 during cardiomyocyte proliferation. The novel splicing mutation c.335-1G > A in NKX2-5 and its potential pathogenic roles in ASD were demonstrated. Our work provides clues not only for deep understanding of NKX2-5 in cardia development, but also for better knowledge in the molecular mechanisms of CHDs.

A case of 46,XY disorders of sex development with congenital heart disease caused by a GATA4 variant.

GATA4 is known to be a causative gene for congenital heart disease, but has also now been associated with disorders of sexual development (DSD). We here report a pathogenic variant of GATA4 in a 46,XY DSD patient with an atrial septal defect, identified by whole-exome sequencing to be c.487C>T (p.Pro163Ser). This mutation resulted in reduced transcriptional activity of the downstream gene. When we compared this transcriptional activity level with other GATA4 variants, those that had been identified in patients with cardiac defects and DSD showed less activity than those in patients with cardiac defect only. This suggests that the normal development of the heart requires more strict regulation of GATA4 transcription than testicular development. Further, when the different variants were co-expressed with wild-type, the transcriptional activities were consistently lower than would be expected from an additive effect, suggesting a dominant-negative impact of the variant via dimer formation of the GATA4 protein. Since these pathogenic GATA4 variants are occasionally identified in healthy parents, a threshold model of quantitative traits may explain the cardiac defect or DSD phenotypes that they cause.

Deciphering complex rearrangements at the breakpoint of an apparently balanced reciprocal translocation t(4:18)(q31;q11.2)dn and at a cryptic deletion: Further evidence of TLL1 as a causative gene for atrial septal defect.

When a de novo balanced reciprocal translocation is identified in patients with multiple congenital abnormalities, attempts are often made to infer the relationship between the phenotype of the patient and genes in the proximity of the breakpoint. Here, we report a patient with intellectual disability, atrial septal defect, syndactyly, and cleft lip and palate who had an "apparently balanced" de novo reciprocal translocation t(4:18)(q31;q11.2) as well as a 7-Mb cryptic deletion spanning the HOXD cluster on chromosome 2q31 that was unrelated to the reciprocal translocation. Further analysis using a nanopore long-read sequencer showed complex rearrangements on both derivative chromosomes 4 and 18 and the deleted chromosome 2. First, the TLL1 locus, which is associated with atrial septal defect, was disrupted by the rearrangement involving chromosome 4. Second, the deleted interval at 2q31 included the entire HOXD cluster, the deletion of which is known to cause toe syndactyly, and the DLX1 and DLX2 loci, which are responsible for cleft lip and palate. Among the haplo-sensitive genes within the deleted interval on 2q31, only the RAPGEF4 gene is known to be associated with an autistic phenotype. Hence, most of the clinical features of the patient could be ascribed to specific genomic rearrangements. We have shown the effectiveness of long-read sequencing in defining, in detail, the likely effects of an apparently balanced translocation and cryptic deletion. The results of the present analysis suggest the possibility of phenotypic prediction through a detailed analysis of structural abnormalities, including balanced translocations and deletions.

TBX5 variant with the novel phenotype of mixed­type total anomalous pulmonary venous return in Holt­Oram Syndrome and variable intrafamilial heart defects.

Variants in T­box transcription factor 5 (TBX5) can result in a wide phenotypic spectrum, specifically in the heart and the limbs. TBX5 has been implicated in causing non­syndromic cardiac defects and Holt­Oram syndrome (HOS). The present study investigated the underlying molecular etiology of a family with heterogeneous heart defects. The proband had mixed­type total anomalous pulmonary venous return (mixed­type TAPVR), whereas her mother had an atrial septal defect. Genetic testing through trio­based whole­exome sequencing was used to reveal the molecular etiology. A nonsense variant was identified in TBX5 (c.577G>T; p.Gly193*) initially showing co­segregation with a presumably non­syndromic presentation of congenital heart disease. Subsequent genetic investigations and more complete phenotyping led to the correct diagnosis of HOS, documenting the novel association of mixed­type TAPVR with HOS. Finally, protein modeling of the mutant TBX5 protein that harbored this pathogenic nonsense variant (p.Gly193*) revealed a substantial drop in the quantity of non­covalent bonds. The decrease in the number of non­covalent bonds suggested that the resultant mutant dimer was less stable compared with the wild­type protein, consequently affecting the protein's ability to bind DNA. The present findings extended the phenotypic cardiac defects associated with HOS; to the best of our knowledge, this is the first association of mixed­type TAPVR with TBX5. Prior to the current analysis, the molecular association of TAPVR with HOS had never been documented; hence, this is the first genetic investigation to report the association between TAPVR and HOS. Furthermore, it was demonstrated that the null­variants reported in the T­box domain of TBX5 were associated with a wide range of cardiac and/or skeletal anomalies on both the inter­and intrafamilial levels. In conclusion, genetic testing was highlighted as a potentially powerful approach in the prognostication of the proper diagnosis.

NKX2-5 variants screening in patients with atrial septal defect in Indonesia.

BACKGROUND: NKX2-5 variant in atrial septal defect patients has been reported. However, it is not yet been described in the Southeast Asian population. Here, we screened the NKX2-5 variants in patients with atrial septal defect (ASD) in the Indonesian population. METHOD: We recruited 97 patients with ASD for genetic screening of the NKX2-5 variant using Sanger sequencing. RESULTS: We identified three variants of NKX2-5: NM_004387.4:c.63A>G at exon 1, NM_004387.4:c.413G>A, and NM_004387.4:c.561G>C at exon 2. The first variant is commonly found (85.6%) and benign. The last two variants are heterozygous at the same locus. These variants are rare (3.1%) and novel. Interestingly, these variants were discovered in familial atrial septal defects with a spectrum of arrhythmia and severe pulmonary hypertension. CONCLUSION: Our study is the first report of the NKX2-5 variant in ASD patients in the Southeast Asian population, including a novel heterozygous variant: NM_004387.4:c.413G>A and NM_004387.4:c.561G>C. These variants might contribute to familial ASD risk with arrhythmia and severe pulmonary hypertension. Functional studies are necessary to prove our findings.