A genome-wide association study in chronic thromboembolic pulmonary hypertension and the ADAMTS13-VWF axis

Chronic thromboembolic pulmonary hypertension (CTEPH) is an important and severe consequence of pulmonary embolism (PE), resulting from failure of thrombus resolution. Identifying genetic risk factors for CTEPH would provide important insights into pathobiology and might allow risk-stratification following PE. A genome-wide association study (GWAS) was performed in 1250 CTEPH patients, 1492 healthy controls and ~7 million single-nucleotide polymorphisms to identify novel disease loci. The ABO locus was identified as the most significant common variant genetic association with CTEPH in both a discovery and validation cohort. The A1 subgroup of ABO was enriched in CTEPH and this may result in multiple functional consequences including variation in plasma von Willebrand factor (VWF) levels. Abnormalities in haemostasis are implicated in CTEPH pathobiology, including elevated levels of VWF, which is cleaved by ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). The ADAMTS13-VWF axis was investigated in 208 CTEPH patients including its relationship to ABO blood groups and ADAMTS13 genetic variants. Plasma ADAMTS13 levels are markedly reduced in CTEPH. This is independent of pulmonary hypertension, disease severity or systemic inflammation. Plasma VWF levels were confirmed to be markedly increased in CTEPH. These findings implicate dysregulation of the ADAMTS13-VWF axis in CTEPH pathobiology

Genetic and Epigenetic Investigations on Pulmonary Hypertension Syndrome in Meat Type- Chickens

This dissertation presents a collection of studies that investigate the genetic and epigenetic associations to ascites phenotype in broiler chickens. Ascites is a significant metabolic disease associated with fast-growing meat-type chickens (broilers) and is a terminal result of pulmonary hypertension syndrome PHS. It is a multi-factorial syndrome caused by interactions between genetic, physiological, environmental, and managemental factors. It was estimated that ascites accounts for losses of about US$1 billion annually worldwide and for over 25% of broilers mortality. Although traditional and molecular genetic methods in the selection and in performance improvements, has greatly reduced ascites frequency, yet it has not eliminated its occurrence. Therefore, this dissertation aimed to 1) develop SNP assays for the gene region of HTR2B to examine the possible association with ascites phenotype and measure gene and allele specific expression in different tissues at different developmental age stages under hypoxic conditions, 2) investigate the association of mitochondrial prevalence in multiple tissues with ascites susceptibility and resistance in broilers, and genes known to regulate mitochondrial biogenesis were assessed, and 3) mapping genome-wide changes in chromatin accessibility for pulmonary artery tissue in ascites - susceptible and ascites- resistant lines under normal and hypoxic conditions using ATAC-seq technology (Assay for Transposase accessible Chromatin with high-throughput sequencing). Altogether, this collection of studies provides new insights into the genetic and epigenetic basis of the ascites syndrome in chicken

Major lung complications of systemic sclerosis (vol 14, pg 511, 2018)

Systemic sclerosis (SSc) is associated with high mortality owing to internal organ complications and lung disease is the leading cause of SSc-associated death. The most notable lung complications in SSc are fibrosis and pulmonary arterial hypertension (PAH). A major challenge for the management of lung disease in SSc is detecting those patients with severe pathology and those patients that are likely to benefit from available treatments. In the past few, strategies for managing lung fibrosis and pulmonary hypertension, including PAH, have greatly progressed. For lung fibrosis, the tools to assess risk of progression and severity of the disease have been refined. Clinical trial results support the use of immunosuppression, including high intensity regimens with autologous stem cell transplantation. New trials are underway to test other potential therapies including treatments that are approved for use in idiopathic lung fibrosis. For PAH, identifying individuals at high risk of disease development is critical. In addition, individuals who have borderline elevation of pulmonary arterial pressure need to be appropriately managed and followed up. Many approved drugs targeting PAH are now available and results from large-scale clinical trials provide robust evidence that various treatments for SSc-associated PAH are associated with good long-term outcomes

Identification of rare sequence variation underlying heritable pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a rare disorder with a poor prognosis. Deleterious variation within components of the transforming growth factor-β pathway, particularly the bone morphogenetic protein type 2 receptor (BMPR2), underlies most heritable forms of PAH. To identify the missing heritability we perform whole-genome sequencing in 1038 PAH index cases and 6385 PAH-negative control subjects. Case-control analyses reveal significant overrepresentation of rare variants in ATP13A3, AQP1 and SOX17, and provide independent validation of a critical role for GDF2 in PAH. We demonstrate familial segregation of mutations in SOX17 and AQP1 with PAH. Mutations in GDF2, encoding a BMPR2 ligand, lead to reduced secretion from transfected cells. In addition, we identify pathogenic mutations in the majority of previously reported PAH genes, and provide evidence for further putative genes. Taken together these findings contribute new insights into the molecular basis of PAH and indicate unexplored pathways for therapeutic intervention

Human genetic determinants of the gut microbiome and their associations with health and disease:a phenome-wide association study

Small-scale studies have suggested a link between the human gut microbiome and highly prevalent diseases. However, the extent to which the human gut microbiome can be considered a determinant of disease and healthy aging remains unknown. We aimed to determine the spectrum of diseases that are linked to the human gut microbiome through the utilization of its genetic determinants as a proxy for its composition. 180 single nucleotide polymorphisms (SNPs) known to influence the human gut microbiome were used to assess the association with health and disease outcomes in 422,417 UK Biobank participants. Potential causal estimates were obtained using a Mendelian randomization (MR) approach. From the total sample analysed (mean age was 57 ± 8 years), 194,567 (46%) subjects were male. Median exposure was 66-person years (interquartile range 59–72). Eleven SNPs were significantly associated with 28 outcomes (Bonferroni corrected P value < 4.63·10−6) including food intake, hypertension, atopy, COPD, BMI, and lipids. Multiple SNP MR pointed to a possible causal link between Ruminococcus flavefaciens and hypertension, and Clostridium and platelet count. Microbiota and their metabolites might be of importance in the interplay between overlapping pathophysiological processes, although challenges remain in establishing causal relationships

Genetics and Genomics of Pulmonary Arterial Hypertension

Pulmonary arterial hypertension is a severe and progressive disorder affecting the blood vessels in the lungs. Typically, symptoms first appear at around 30–40 years of age and, without treatment, can lead to fatal heart disease within a few years. Genetic studies over the past decade have identified numerous genes that contribute to disease progression but, for many sufferers, the underlying genetic cause remains elusive. The collection of reviews and original research articles contained within this book provide an overview of recent advancements in understanding the genetic risk factors for pulmonary arterial hypertension. We further examine the emerging interplay between genetic variants and clinical outcomes, providing a framework for new treatments and improved patient care

Characterising ATP13A3 biological function and its role in pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is a rare but devastating disease characterised by the progressive remodelling of the small pulmonary vessels. Although the causes may vary, similar pathobiological features are shared among different forms of PAH, with endothelial dysfunction, the hyperproliferation of smooth muscle cells and mesenchymal cells in the vascular wall, as well as inflammation contributing to this process. Despite the availability of licensed therapies, 5-year survival for patients remains less than 60%. To uncover additional fundamental mechanisms of PAH pathobiology, our group undertook whole genome sequencing in 1038 idiopathic or heritable PAH patients and identified disease-associated heterozygous mutations in ATP13A3, a P5B-type ATPase. P5 ATPases, namely ATP13A1-5, are putative transmembrane proteins that generate and maintain significant chemical gradients across biological membranes by active transport of cations. However, to date, studies of their functions are sparse. Therefore, the focus of my project was to characterise ATP13A3 in the pulmonary vasculature and to uncover the underlying mechanism of how mutations in this gene contribute to PAH pathogenesis. Although the biological functions of ATP13A3 are elusive, its homologue (CATP-5) in C. elegans was previously reported as a polyamine transporter. Polyamines are a group of naturally existing polycations essential for many cellular processes. Therefore, my working hypothesis was that ATP13A3 loss of function might disrupt the polyamine homeostasis and hence contribute to the abnormal vascular remodelling in PAH. To address this, I first confirmed ATP13A3 as residing in the recycling endosome of endothelial cells. This localisation of ATP13A3 suggested it might be related to polyamine transport since the endocytic pathway was recognised as a crucial route for polyamine trafficking. To expand this finding, I developed molecular tools to knockdown or to overexpress ATP13A3 in vascular cells and analysed how these affected polyamine transport. This led to the discovery of putrescine as the preferred substrate for ATP13A3 and also revealed a basal reduction of other polyamines in endothelial cells. Further investigation highlighted an essential role of ATP13A3 in pulmonary vasculature with its deficiency restrained cell growth and predisposed endothelial cells to apoptosis. To validate these findings, blood outgrowth endothelial cells (BOECs) were isolated from a PAH patient bearing an LK726X ATP13A3 frameshift mutation (LK726X). Intriguingly, ATP13A3 causing ATP13A3 reduction, impaired polyamine uptake and induced apoptosis in BOECs. This provides the first evidence for the pathogenic effects of a PAH associated ATP13A3 mutation. In addition, using a lentiviral overexpression system, I also demonstrated impairments of other PAH- associated mutations on ATP13A3 mediated polyamine uptake, reinforcing the involvement of ATP13A3 in PAH pathobiology. To explore whether ATP13A3 mutations can cause PAH, our laboratory has generated a mouse model harbouring a human ATP13A3 mutation (P452Lfs) via the MRC Harwell Gene editing service. My colleague, Dr Ekaterina Legchenko and I have discovered that the Atp13a3P452Lfs/P452Lfs mice spontaneously develop PAH-like hemodynamic changes at 6-months of age. Preliminary data also revealed lower Atp13a3 level and reduced polyamine content in the lungs of the mutant mice, suggesting that Atp13a3 frameshift mutation (P452L) may contribute to PAH pathogenesis via the disruption of polyamine homeostasis in vivo. In conclusion, my work characterises for the first time that ATP13A3 is a polyamine transporter in vascular cells with putrescine as its preferable transport substrate. ATP13A3 mutations identified from PAH patients are highly likely to impair polyamine homeostasis and contribute to PAH progression. These findings open up a new field for investigating PAH pathobiology and also highlight the potential role of rebalancing polyamine homeostasis in PAH treatment

Avian Cardiovascular Disease Characteristics, Causes and Genomics

Cardiovascular disease is common in avian species and increasing commercial economic losses and demand for healthcare in the household/smallholding veterinary sector has resulted in increased research into these disorders. This in turn has highlighted the importance of breeding, genetic testing and possibilities for future prognostic and diagnostic testing. Research into avian cardiovascular genetics has rapidly accelerated. Previously much work was undertaken in mammals with information extrapolated and transferred to birds. Birds have also been used to model cardiovascular disease and therefore knowledge has become enriched due to this endeavour. Increasingly, the avian genome is being analysed in its own right. This work is assisted by the growing number of avian genomes being published. In 2015, Nature published news on the ‘Bird 10K’ project, which aims to sequence 10,500 extant bird species. By 2018, the Avian Genomes Consortium had published the sequences of 45 species/34 orders. This review investigates a range of avian cardiovascular disorders in order to highlight their pathologies, epidemiology and genetics in addition to avian models of heart disease. With the availability of more reference genomes, increases in the number and magnitude of avian studies and more advanced technologies, the genetics behind avian cardiovascular disorders is being unravelled

Thoracic aortic aneurysms and dissections: genetic analysis of Mendelian and complex cases

The present doctoral thesis deals with the still partially unraveled genetic component of thoracic aortic aneurysms and dissections, a frequently asymptomatic but potentially lethal condition and major cause of sudden death. Our main objective was to contribute to further elucidate the genetics behind it, from both Mendelian and complex perspectives. We analyzed single and familial, forensic and clinical mendelian cases applying either a candidate-gene or whole exome massive parallel sequencing approach, respectively. We were able to solve approximately 23% of the forensic single cases and identified two strong candidate mutations in TGFB2 and PRKG1 genes in the two non-syndromic familial cases analyzed. For the analysis of complex cases we chose a population-based approach. We selected bicuspid aortic valve patients with and without concomitant thoracic aortic dilation and faced them against general population controls. We were not able to identify any consistently significant association, though a promising one arose involving HMCN2 and calcium metabolism that should be considered in future studies. The direct clinical consequences some of these results had supported molecular diagnosis, reliable genotype-phenotype correlations, and risk stratification as important tools for clinical management of these patients and family members at risk, as well as the need of research to continue