Molecular Function and Contribution of TBX4 in Development and Disease.

Over the past decade, recognition of the profound impact of the TBX4 (T-box 4) gene, which encodes a member of the evolutionarily conserved family of T-box-containing transcription factors, on respiratory diseases has emerged. The developmental importance of TBX4 is emphasized by the association of TBX4 variants with congenital disorders involving respiratory and skeletal structures; however, the exact role of TBX4 in human development remains incompletely understood. Here, we discuss the developmental, tissue-specific, and pathological TBX4 functions identified through human and animal studies and review the published TBX4 variants resulting in variable disease phenotypes. We also outline future research directions to fill the gaps in our understanding of TBX4 function and of how TBX4 disruption affects development.

Human Genetics of Semilunar Valve and Aortic Arch Anomalies.

Lesions of the semilunar valve and the aortic arch can occur either in isolation or as part of well-described clinical syndromes. The polygenic cause of calcific aortic valve disease will be discussed including the key role of NOTCH1 mutations. In addition, the complex trait of bicuspid aortic valve disease will be outlined, both in sporadic/familial cases and in the context of associated syndromes, such as Alagille, Williams, and Kabuki syndromes. Aortic arch abnormalities particularly coarctation of the aorta and interrupted aortic arch, including their association with syndromes such as Turner and 22q11 deletion, respectively, are also discussed. Finally, the genetic basis of congenital pulmonary valve stenosis is summarized, with particular note to Ras-/mitogen-activated protein kinase (Ras/MAPK) pathway syndromes and other less common associations, such as Holt-Oram syndrome.

Defining the clinical validity of genes reported to cause pulmonary arterial hypertension.

PURPOSE: Pulmonary arterial hypertension (PAH) is a rare, progressive vasculopathy with significant cardiopulmonary morbidity and mortality. Genetic testing is currently recommended for adults diagnosed with heritable, idiopathic, anorexigen-, hereditary hemorrhagic telangiectasia-, and congenital heart disease-associated PAH, PAH with overt features of venous/capillary involvement, and all children diagnosed with PAH. Variants in at least 27 genes have putative evidence for PAH causality. Rigorous assessment of the evidence is needed to inform genetic testing. METHODS: An international panel of experts in PAH applied a semi-quantitative scoring system developed by the NIH Clinical Genome Resource to classify the relative strength of evidence supporting PAH gene-disease relationships based on genetic and experimental evidence. RESULTS: Twelve genes (BMPR2, ACVRL1, ATP13A3, CAV1, EIF2AK4, ENG, GDF2, KCNK3, KDR, SMAD9, SOX17, and TBX4) were classified as having definitive evidence and 3 genes (ABCC8, GGCX, and TET2) with moderate evidence. Six genes (AQP1, BMP10, FBLN2, KLF2, KLK1, and PDGFD) were classified as having limited evidence for causal effects of variants. TOPBP1 was classified as having no known PAH relationship. Five genes (BMPR1A, BMPR1B, NOTCH3, SMAD1, and SMAD4) were disputed because of a paucity of genetic evidence over time. CONCLUSION: We recommend that genetic testing includes all genes with definitive evidence and that caution be taken in the interpretation of variants identified in genes with moderate or limited evidence. Genes with no known evidence for PAH or disputed genes should not be included in genetic testing.

First Genotype-Phenotype Study in TBX4 Syndrome: Gain-of-Function Mutations Causative for Lung Disease.

Rationale: Despite the increased recognition of TBX4 (T-BOX transcription factor 4)-associated pulmonary arterial hypertension (PAH), genotype-phenotype associations are lacking and may provide important insights. Objectives: To compile and functionally characterize all TBX4 variants reported to date and undertake a comprehensive genotype-phenotype analysis. Methods: We assembled a multicenter cohort of 137 patients harboring monoallelic TBX4 variants and assessed the pathogenicity of missense variation (n = 42) using a novel luciferase reporter assay containing T-BOX binding motifs. We sought genotype-phenotype correlations and undertook a comparative analysis with patients with PAH with BMPR2 (Bone Morphogenetic Protein Receptor type 2) causal variants (n = 162) or no identified variants in PAH-associated genes (n = 741) genotyped via the National Institute for Health Research BioResource-Rare Diseases. Measurements and Main Results: Functional assessment of TBX4 missense variants led to the novel finding of gain-of-function effects associated with older age at diagnosis of lung disease compared with loss-of-function effects (P = 0.038). Variants located in the T-BOX and nuclear localization domains were associated with earlier presentation (P = 0.005) and increased incidence of interstitial lung disease (P = 0.003). Event-free survival (death or transplantation) was shorter in the T-BOX group (P = 0.022), although age had a significant effect in the hazard model (P = 0.0461). Carriers of TBX4 variants were diagnosed at a younger age (P < 0.001) and had worse baseline lung function (FEV1, FVC) (P = 0.009) than the BMPR2 and no identified causal variant groups. Conclusions: We demonstrated that TBX4 syndrome is not strictly the result of haploinsufficiency but can also be caused by gain of function. The pleiotropic effects of TBX4 in lung disease may be in part explained by the differential effect of pathogenic mutations located in critical protein domains.

ADA2 deficiency complicated by EBV-driven lymphoproliferative disease.

A 29-year old male with recurrent respiratory and skin infections, anaemia and neutropaenia during childhood required immunoglobulin replacement for antibody deficiency from age 16. He remained relatively well until age 28 when he presented with a two-week history of fatigue, sore throat, fever and productive cough. He was found to have EBV viraemia and splenomegaly and a diagnosis of EBV-driven lymphoproliferative disease was made following bone marrow trephine. Family history was notable with three siblings: a healthy sister and two brothers with anaemia and neutropaenia; one who succumbed to septicaemia secondary to neutropaenic enterocolitis age 5 and another who developed intestinal vasculitis and antibody deficiency and had a successful haemopoetic stem cell transplant. The proband's DNA underwent targeted sequencing of 279 genes associated with immunodeficiency (GRID panel). The best candidates were two ADA2 variants, p.Arg169Gln (R169Q) and p.Asn370Lys (N370K). Sanger sequencing and co-segregation of variants in the parents, unaffected sister and all three affected brothers was fully consistent with compound heterozygous inheritance. Subsequent whole genome sequencing of the proband identified no other potential causal variants. ADA2 activity was consistent with a diagnosis of ADA2 deficiency in affected family members. This is the first description of EBV-driven lymphoproliferative disease in ADA2 deficiency. ADA2 deficiency may cause susceptibility to severe EBV-induced disease and we would recommend that EBV status and viral load is monitored in patients with this diagnosis and allogeneic SCT is considered at an early stage for patients whose ADA2 deficiency is associated with significant complications.

Identification of novel pathogenic variants and features in patients with pseudohypoparathyroidism and acrodysostosis, subtypes of the newly classified inactivating PTH/PTHrP signaling disorders.

Albright hereditary osteodystrophy (AHO) is a complex disorder defined by the presence of a short adult stature relative to the height of an unaffected parent and brachydactyly type E, as well as a stocky build, round face, and ectopic calcifications. AHO and pseudohypoparathyroidism (PHP) have been used interchangeably in the past. The term PHP describes end-organ resistance to parathyroid hormone (PTH), occurring with or without the physical features of AHO. Conversely, pseudopseudohypoparathyroidism (PPHP) describes individuals with AHO features in the absence of PTH resistance. PHP and PPHP are etiologically linked and caused by genetic and/or epigenetic alterations in the guanine nucleotide-binding protein alpha-stimulating (Gs α) locus (GNAS) in chromosome 20q13. Another less-recognized group of skeletal dysplasias, termed acrodysostosis, partially overlap with skeletal, endocrine, and neurodevelopmental features of AHO/PHP and can be overlooked in clinical practice, causing confusion in the literature. Acrodysostosis is caused by defects in two genes, PRKAR1A and PDE4D, both encoding important components of the Gs α-cyclic adenosine monophosphate-protein kinase A signaling pathway. We describe the clinical course and genotype of two adult patients with overlapping AHO features who harbored novel pathogenic variants in GNAS (c.2273C > G, p.Pro758Arg, NM_080425.2) and PRKAR1A (c.803C > T, p.Ala268Val, NM_002734.4), respectively. We highlight the value of expert radiological opinion and molecular testing in establishing correct diagnoses and discuss phenotypic features of our patients, including the first description of subcutaneous ossification and spina bifida occulta in PRKAR1A-related acrodysostosis, in the context of the novel inactivating PTH/PTH related peptide signaling disorder classification system.

'There and Back Again'-Forward Genetics and Reverse Phenotyping in Pulmonary Arterial Hypertension.

Although the invention of right heart catheterisation in the 1950s enabled accurate clinical diagnosis of pulmonary arterial hypertension (PAH), it was not until 2000 when the landmark discovery of the causative role of bone morphogenetic protein receptor type II (BMPR2) mutations shed new light on the pathogenesis of PAH. Since then several genes have been discovered, which now account for around 25% of cases with the clinical diagnosis of idiopathic PAH. Despite the ongoing efforts, in the majority of patients the cause of the disease remains elusive, a phenomenon often referred to as "missing heritability". In this review, we discuss research approaches to uncover the genetic architecture of PAH starting with forward phenotyping, which in a research setting should focus on stable intermediate phenotypes, forward and reverse genetics, and finally reverse phenotyping. We then discuss potential sources of "missing heritability" and how functional genomics and multi-omics methods are employed to tackle this problem.

The use of panel testing in familial breast and ovarian cancer.

Advances in sequencing technology have led to the introduction of panel testing in hereditary breast and ovarian cancer. While direct-to-consumer testing services have become widely available, the clinical validity of many of the genes on panel tests remains contentious and risk management guidelines are often lacking. This article gives an overview of advantages with panel testing as well as important challenges, including clinical translation of test results.

Tachycardia-induced cardiomyopathy in pregnancy.

Heart failure in pregnancy is rare, but usually ascribed to peripartum cardiomyopathy in the absence of other possible diagnoses. However, heart failure can develop solely due to a tachycardia, so-called 'tachycardia-induced cardiomyopathy'. The incidence of tachycardia-induced cardiomyopathy in pregnancy is unknown, but it is a treatable and potentially reversible cause of heart failure. Clinically, tachycardia-induced cardiomyopathy during pregnancy might present in a similar manner, but its management has to be individualized according to the arrhythmic substrate and usually involve multidisciplinary input from specialists in obstetrics, cardiac electrophysiology and heart failure.

Histopathology of the great vessels in patients with pulmonary arterial hypertension in association with congenital heart disease: large pulmonary arteries matter too.

BACKGROUND: Pulmonary arterial hypertension (PAH) is considered primarily a disease of the distal pulmonary arteries whereas little is known on the effect of long-standing pulmonary hypertension on the larger proximal pulmonary arteries. This study aims to investigate the structural changes in the great arteries of adults who developed PAH in association with congenital heart disease (CHD), with severe cases termed Eisenmenger syndrome. METHODS: We performed macroscopic and light microscopy analyses on the great arteries of 10 formalin-fixed human hearts from patients with PAH/CHD and compared them to age-matched healthy controls. A detailed histology grading score was used to assess the severity of medial wall abnormalities. RESULTS: Severe atherosclerotic lesions were found macroscopically in the elastic pulmonary arteries of 4 PAH/CHD specimens and organised thrombi in 3; none were present in the controls. Significant medial wall abnormalities were present in the pulmonary trunk (PT), including fibrosis (80%), and atypical elastic pattern (80%). Cyst-like formations were present in less than one third of patients and were severe in a single case leading to wall rupture. The cumulative PT histology grading score was significantly higher in PAH/CHD cases compared to controls (p<0.0001) and correlated positively with larger PT diameters (ρ=0.812, p<0.0001) and the degree of medial wall hypertrophy (ρ=0.749, p<0.0001). CONCLUSIONS: Chronic PAH in association with CHD results in marked macroscopic and histological abnormalities in the large pulmonary arteries. These abnormalities are likely to affect haemodynamics and contribute to morbidity and mortality in this cohort.

Detrimental impact of socioeconomic status on exercise capacity in adults with congenital heart disease.

OBJECTIVES: To evaluate the relationship between socioeconomic status (SES), access to physical activity resources, urban-rural dwelling, levels of pollution and exercise capacity in adult congenital heart disease (ACHD) patients. BACKGROUND: Exercise intolerance is prevalent in ACHD and the contributing factors are poorly understood. METHODS: A total of 1268 ACHD patients living in England who underwent cardiopulmonary exercise testing at our center were included. Neighborhood deprivation (English Indices of Deprivation), urban-rural dwelling, availability of green space, distance to the closest gym/fitness center and levels of pollution were estimated based on administrative data. RESULTS: Urban-rural dwelling, availability of green space and levels of pollution were unrelated to exercise capacity. Lower SES was associated with a significantly lower peak oxygen consumption (P<0.002) and heart rate reserve (P<0.004). This association was non-linear and most pronounced in ACHD patients with cardiac defects of medium complexity living in the most socioeconomically disadvantaged communities. Low SES was associated with higher prevalence of diabetes (P=0.015) and smoking (P=0.01). Coronary artery disease was rare in this young population and low SES was found to be related to exercise capacity independently of the presence of coronary artery disease. CONCLUSIONS: Living in poorer areas is associated with exercise intolerance in contemporary ACHD patients. Although low SES is linked to traditional cardiovascular risk factors, the deleterious effects of SES on exercise capacity seem to be only partially mediated via coronary artery disease. Reducing social inequalities in ACHD patients may have a positive effect on quality of life and long-term prognostic implications.

Peak oxygen uptake correlates with disease severity and predicts outcome in adult patients with Ebstein's anomaly of the tricuspid valve.

BACKGROUND: Ebstein's anomaly of the tricuspid valve often results in biventricular dysfunction and functional deterioration. Little is known about the relation between exercise capacity, disease severity and outcome in adults with Ebstein's anomaly. METHODS: Data on all patients with Ebstein's anomaly of the tricuspid valve who underwent cardiopulmonary exercise testing in our tertiary center were collected. The relation between exercise parameters, anatomic severity (Glasgow outcome score) and the combined end-point of death, non-elective hospitalization and surgical repair was studied using Cox regression analysis. RESULTS: A total of 51 adult patients fulfilled inclusion criteria (49% male, mean age 37.8±13.6 years). Mean peak oxygen uptake (peak VO2) was 63.2±18.7% of predicted, the slope of ventilation per unit of carbon dioxide output (VE/VCO2 slope) 37.4±11.4, heart rate reserve (HRR) 23.6±22.7 bpm. A significantly lower peak VO2 was found in patients with a higher Glasgow outcome score, higher cardiothoracic ratio and documented atrial shunt. Peak VO2 (HR for value <60% of predicted 3.47, 95% CI: 1.28­9.44, p=0.015) and HRR (HR for value <25 bpm 3.07, 95% CI: 1.24­7.61, p=0.016) were significant predictors of outcome, the former being the strongest on multivariable analysis. CONCLUSIONS: Reduced exercise capacity in patients with Ebstein's anomaly relates to severity of the underlyingdisease and is a strong and independent predictor of outcome. Cardiopulmonary exercise testing should be incorporated in the follow-up and risk stratification of patients with this relatively uncommon and challenging cardiac defect.

Exercise intolerance in patients with congenitally corrected transposition of the great arteries relates to right ventricular filling pressures.

BACKGROUND: Patients with congenitally corrected transposition of the great arteries (ccTGA) have significantly reduced exercise tolerance. Progressive right ventricular (RV) dysfunction with tricuspid regurgitation (TR) and other haemodynamic lesions are common among them. We hypothesised that interaction of these factors may result in increased systemic RV filling pressure, which in turn impact on exercise capacity. METHODS: Patients with ccTGA in functional class I or II, able to perform treadmill exercise and without resting cyanosis were enrolled. All patients underwent cardiopulmonary exercise testing and transthoracic echocardiographic examination. RV filling pressure was estimated using tissue Doppler imaging (TDI) techniques by measuring early annular diastolic velocity (Ea) and the ratio of the transtricuspid inflow to the early annular diastolic velocity (E/Ea). RESULTS: A total of 27 patients (mean age 41 years, 48% female) were assessed, the majority (63%) asymptomatic. Many patients had coexistent haemodynamic lesions including shunts, pulmonary stenosis, TR and systemic ventricular dysfunction. Average percentage predicted peak oxygen consumption, VE/VCO(2) slope and heart rate reserve were abnormal in this population. Patients with moderately/severely impaired exercise capacity (≤ 60% predicted peak VO(2)) had significantly higher E/Ea ratios compared to those with normal/mildly impaired exercise capacity (septal E/Ea = 17.1 ± 9.7 vs 8.8 ± 1.6 and lateral E/Ea = 11.5 ± 5.8 vs 6.6 ± 1.3, p = 0.007 and 0.01 respectively). CONCLUSION: Reduced exercise capacity is common in adults with ccTGA even among asymptomatic patients and relates to increased RV filling pressures assessed by TDI. This index could potentially be used to optimize therapy or prognosticate adverse events in ccTGA patients.