Modifiers of elastin arteriopathy

National Heart, Lung, And Blood Institute

Investigators

Linked publications, trials & patents

Abstract

To characterize genetic variation that impacts the severity of blood vessel disease in patients with elastin insufficiency and Williams syndrome, we are using a combination of approaches. First, using a technique called quantitative trait locus analysis, we identified different regions of the mouse genome where differences in genetic background impact the degree of hypertension and vascular narrowing in Eln+/- mice. Building on data from previous years, we found that NCF1, a component of the NADPH oxidase system, is important for generating reactive oxygen species (ROS) in elastin insufficient vessels. Its production is triggered by the increased shear stress seen in Eln+/- arteries and, once produced, contributes to the high blood pressure and vascular stiffness observed in these patients and animals. Inhibition of ROS production using chemical or genetic modifications, decreases blood pressure and stiffness. In the last year, we completed evaluation of vascular reactivity differences in this model and were able to publish the work (Function). Our previous modifier studies in human identified new pathways, such as the adaptive immune system that might modify phenotypic outcome for patients with WS. To model this, we previously bred the Eln+/- mouse to the Rag1-/-. The Rag1-/- mouse lacks B and T cells and, when present in the Eln+/- background, the Eln+/- mice have improved caliber and normalized blood pressure. Unfortunately, the Eln+/- mouse does not mimic the focal stenosis commonly seen in people with WS, mimicking more the long segment stenosis seen in this population. We hypothesize though that while elastin haploinsufficiency predisposes to both features, that different modifier pathways are responsible for driving the two phenotypes. As such, we have begun work on a new Elnfl/fl model which, when bred to a smooth muscle cre, produces more focal stenosis with a notable inflammatory component. We are in the process of breeding the Rag1-/- mouse to this new background to test whether limiting the inflammatory response improves or exacerbates the stenosis phenotype. Also on the mouse side, we have begun investigation of a second mouse line that contains a knock-out mutation in one of the other genes in the WS locus. This gene, Baz1b, is a widely expressed transcription factor thought to predominantly impact cells derived from the neural crest. The knockout mice are born alive but generally die within 24 hoursthe cause of which is still being investigated. The heterozygous mice do have reduced survival relative to WT mice and have slower growth. Current efforts involve analysis of the cause of sudden death in this line. Previous, now retracted, studies by other investigators reported evidence of developmental cardiac anomalies such as tetalology of Fallot and coarctation in a different Baz1b line due to the impact of the gene on neural crest. Our studies to date have not identified a clear cardiac phenotype although, decreased systolic function is seen relative to WT. We have also begun investigating the impact of combined variation between elastin and Baz1b in this model as well as its impact on growth, lung and GI function. On the human side of our investigations, we have expended our previous whole exome sequencing project into the largest whole genome sequencing project on Williams Syndrome to date, with collaboration of scientists in Italy, Canada and the United States. We are continuing to look at the association of rare and common genomic events in 3 levels (single nucleotide variants (SNVs), structural variants (SV), copy number variants (CNVs)) with vascular disease severity in people with WS. Recently, we performed whole genome sequencing on 188 subjects with WS at from NIH, 65 from Telethon Biobank, Italy, 248 from the University of Toronto, and 10 from Boston Childrens Hospital at the Uniformed Services University of Health Science. We have done alignment of reads and joint variant callings using GATK on the NIH biowulf cluster, and imputed/phased single nucleotide variants using the TOPMED imputation server. We have completed SNVs and CNVs callings and completed about 70-80% SVs callings. With these data in hand, we looked at the association of SVAS severity with non-synonymous SNVs. Using new methods, aimed at identifying pathways with increased frequency of pathogenic variant frequency in people with severe vs no stenosis, we have confirmed the interesting pathways identified in our exome study, including the matrisome, immune and MTOR/PIK3CA pathways. We are currently working on a manuscript. Additionally, we have been working on CNV analysis of the WS subjects. Analysis of CNVs in short read genomes is relatively new and before comparing severe vs no stenosis subgroups as done in our modifier study, we want to confirm the global impact of WS on the genome by comparing CNV frequency in people with WS vs controls. Work to date has identified secondary deletions in indviduals with WS which we are now validating using a variety of methods (chromosomal microarray, copy number analyis and long read DNA sequencing depending on the specific CNV identified. Such efforts may allow us to call WS deletion boundaries with greater certainty (a task that is currently difficult due to the redundancy of the genome in those areas). In that way, deletion size can be better assessed for its impact on phenotypic outcomes. In collaboration with Max Muenke and Paul Kruszka, formerly of NHGRI, we completed an epidemiological study in 52 mothers of individuals with WBS. In this study, our WBS patients initially served as a control group to his holoprosencephaly (HPE) cohort. A second manuscript looking at environmental influences that impact disease severity was published this year (Birth Defects Res). In addition, we separately analyzed data looking for differences in vascular outcomes based on pre- and perinatal exposures within the WBS cohort. We again found a signal for the immune system in the form of maternal allergy. Before initiating a protocol for the collection of biospecimens, we are seeking to confirm this phenotype on a larger cohort and as such, have added additional perinatal exposure questions to our DNA and Tissue Bank. In both the Bank questionnaire and a second new study aimed at identifying the impact of COVID in a host of rare diseases affecting the vasculature, lungs and immune system, we have begun asking questions about COVID exposures and outcomes, as well as response to vaccination. Given the known morbidities associated with this condition, we anticipate that exposure and experience with this disease may influence outcomes about which we have not yet become aware. Recording this information now will allow us to follow these features in our patients as part of their longterm surveillance. Taken together, these complimentary projects will allow us to identify important pathways that interact with elastin insufficiency to alter disease severity. The discovery of modifier pathways may direct us to novel therapies to improve vascular health. Because elastin concentration decreases with age, the lessons learned from these rare disease patients have the potential to provide information regarding genes and pathways that impact blood vessels in aging individuals. Separate from the WS work, we have also collaborated with geneticists around the globe to improve the current system for naming of rare conditions. Our proposed approach includes using both the gene name and phenotype in the dyadic description. The association between the genes and phenotypes are increasingly important as we move closer to the time when precision medicine approaches will be used to improve health for individuals based on their molecular signature.