Monogenetic disorders grant the unique prospect to understand complex diseases due to a single defined genetic defect. Advanced sequencing technology accelerates the discovery of gene candidates, but progress is hampered by the inability to distinguish between functional disease related and non-disease related mutations. Patient-specific in-vitro disease modeling systems are used in combination with genetic tools to identify disease related mutation and provide the ability to study gene related disease mechanism and to perform drug screening on an individual level. Arterial calcifications contribute to morbidity and are predictors of premature death. Vascular calcification is presented as a secondary complication to diseases such as atherosclerosis, diabetes mellitus type II, and chronic kidney disease. The mechanism of this pathology is poorly understood. We identified a novel genetic disease in which de novo vascular calcifications form in the lower extremity arteries of affected adults in a consanguineous family. A single nucleotide polymorphism array revealed a shared 22.4 MB region of homozygosity on chromosome 6 in all affected members. Direct sequencing identified a homozygous nonsense mutation (c662C>A; pS221X) in NT5E, encoding CD73, an enzyme involved in the extracellular purine metabolic pathway. CD73 converts extracellular AMP to adenosine and inorganic phosphate. Cultured fibroblasts isolated from affected individuals show complete loss of CD73 enzyme activity and that these cells display increased tissue non-specific alkaline phosphatase (TNAP) activity, a key enzyme necessary for calcification. Genetic rescue and treatment with adenosine reduced both TNAP activity and in vitro calcification in the patient fibroblasts, suggesting that adenosine signaling in normal tissue inhibits ectopic vessel calcification. Adenosine signaling has been shown to be protective during harmful events in the cardiovascular system, however the link between adenosine and protection from calcification is unprecedented. This vascular phenotype shares calcification pathologies seen in diabetes mellitus and end-stage renal failure. We are investigating if adenosine or other members of the extracellular purine metabolic pathways modulates heterotopic calcifications in potentially related disorders like, Pseudoxanthoma elastic, Fahr Syndrome, Pseudohypoparathyreodism type1a. Prolidase deficiency (PD) is a rare autosomal recessive disorder characterized by recurrent lower extremity ulcerations, recurrent infections, telangiectasias, and mild developmental delays. Prolidase, the defective peptidase, is responsible for the breakdown of proline and hydroxyproline dipeptides, thus playing a major role in collagen degradation and recycling of proline. The pathogenesis is not well understood, therapy is mainly supportive, and prognosis is poor. Case reports of PD speculate it as a cause of Hyper IgE syndrome (HIES), as similarities exist between the two conditions that are caused by mutations in STAT3. Both show elevated serum IgE, pulmonary infections often resulting in parenchymal damage such as bronchiectasis, hyperextensibility and similar facial features. Like PD, the pathogenesis of many HIES features remain poorly understood. Both PD and HIES appear to have abnormalities with wound healing, manifested primarily by the pronounced skin ulcerations in PD and abnormal lung healing after pneumonias and lung surgeries in HIES. Our preliminary studies show defects in extracellular matrix degrading enzymes MMP 1,3, 9, 12 and proangiogenic factors like FLK1, VEGF, and Endothelin in PD and HIES fibroblasts after stimulation with TNF-alpha. These abnormalities are thought to involve dysregulated HIF signaling. In vitro, the MMP and angiogenic defects in PD and HIES fibroblasts improve after supplementation with hydroxyproline. Further studies with more patient samples are needed to define the mechanism and patient-patient variability and to further explore hydroxyproline as a potential therapy for these individuals. Turner syndrome (TS) is the most common genetic disorder affecting females and encompasses a broad spectrum of features, most notably short stature and loss of ovarian function. results from loss of all or part of one sex chromosome. Individual genetic mutations can alter different metabolic pathways ultimately affecting multiple tissue and or cellular functions. A cardiovascular phenotype is well established in the TS population, which presents with cardiac valve abnormalities. Advanced imaging revealed an additional set of structural cardiovascular abnormalities, including the aortic arch and associated with high incidence of aortic dissection. Additionally, pathological changes in the aortic arch resemble Marfan syndrome, caused by a mutation in fibrillin1 and associated with increased TGF-b activity. We are recruiting TS patients and generating patient specific cell lines for further functional studies. Several autoinflammatory diseases, such as SAVI, DADA2, NOMID and CANDLE, negatively impact the health of blood vessels and can result in severe tissue damage as well as fatal outcomes for the subjects affected. These diseases are often caused by genetic mutations but the underlying mechanisms have not been well characterized. We are investigating and comparing the genetic characteristics of these diseases through clinical evaluation, genetic testing to include whole genome sequencing, high content screening and single cell sequencingas well as patient derived cells (myeloid) to investigate the disease mechanisms in vitro and in vivo murine models. Small vessel diseases are conditions characterized by the narrowing of small arteries leading to an imbalance of blood supply upon demand, resulting in progressive chronic hypoperfusion with detrimental outcomes. CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarct and leukoencephalopathy) is caused by mutations in NOTCH3. The disease is of slow onset, with initial clinical manifestations in the third and fourth decade of life, but progressive and fatal with no available cure. Predominant clinical features include migraine with aura (atypical or isolated), strokes, memory loss, and multiple psychiatric symptoms including dementia. Similarly, Degos disease is a poorly understood rare genetic disorder leading to defects in coagulation, in which small veins and arteries become occluded. Initial symptoms present as skin lesions for both the benign and systemic forms of the disease. However, benign Degos is limited to skin lesions, while systemic Degos progresses to other organ systems (intestines, CNS) and becomes fatal within 2-3 years of diagnosis. We are working to characterize the etiology and natural history of CADASIL and Degos patients through comprehensive clinical and molecular characterizations.
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