Genome-wide Epistasis for Cardiovascular Severity in Marfan Study

Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder with pleiotropic manifestations in the ocular, skeletal and cardiovascular systems. Morbidity and mortality are mostly determined by aortic root aneurysm dissection and rupture. Although mutations in FBN1, the gene coding for the extracellular matrix protein fibrillin-1, are the well-established genetic cause of this condition, there is a very poor correlation between the nature or location of the causal FBN1 mutation and the phenotypical outcome. Indeed, wide intra- and interfamilial phenotypical variability is observed. So, even with an identical primary mutation in all family members, the clinical spectrum varies widely, from completely asymptomatic to sudden death due to aortic dissection at a young age. The precise mechanisms underlying this variability remain largely elusive.

Consequently, a better understanding of the functional effects of the primary mutation is highly needed and the identification of genetic variation that modifies these effects is becoming increasingly important. In this project, we have carefully selected different innovative strategies to discover mother nature's own modifying capabilities with respect to Marfan syndrome aortopathy.

In this project we will focus on the cardiovascular, or more specific, the TAAD (Thoracal Aorta Aneurysma Dissection) expressivity of the Marfan syndrome. The most frequent mutations in FBN1 (fibrilin-1 ), with significant aortopathy expressivity is p.Ile2585Thr; c.7754T\>C and p.Ala882Val; c.2645C\>T). We will limit the used population to p.Ile2585Thr; c.7754T\>C mutation since this is the biggest population.

Marfan syndrome subjects carrying an identical FBN1 mutation show a variable aortopathy expressivity, even within one family. We hypothesize that the cardiovascular phenotypical variability is under control of genetic modifiers.

The first approach strategy involves ranking of carriers of the specific FBN1 mutation that present with significant variable aortopathy expressivity according to the severity of aortic aneurysma disease (based on Z-score, timing of surgery and manual expert curation). We will stratify these mutation carrying individuals in three groups: mild or no aortic disease (UMC, unaffected mutation carrier)), severely affected (AMC, affected mutation carrier), and participants with indeterminate data.

The second approach is the molecular characterisation of the 25% extreme cohort (AMC and UMC) using WGS (Whole Genome Sequencing) and linkage analysis.

Finally subjects peripheral blood mononuclear cells (PBMCs) of 10 severely affected mutation carrier (AMC) and 10 unaffected mutation carriers (UMC) as well as 2 controls will be reprogrammed to iPSCs (induced Pluripotential Stem Cells). These cells will finally be differentiated into VSMC's (VasculairSmoth Muscle Cells). The genomic integrity and identity of the iPSCs and the VSMCs will be validated using RT-PCR and immunocytochemistry.

Transcriptomic (i.e. RNA-sequencing) data will be acquired from these specific induced pluripotent stem cell-derived vascular smooth muscle cells (iPSC-VSMCs).

We will be able to filter the WGS data based on variant quality and location in genes that are differentially expressed when comparing the AMC and UMC iPSC-VSMCs, via the synchronization of both data types. This approach will allow us to identify the modifier gene. Once candidate modifier genes (and hence candidate modifier variants) have been identified, their modifying capacity will be functionally checked in relevant cell- or animal models. The choice of the model system will be determined based on the nature of the identified modifier. In an animal model, we will prove its effect by crossing an animal carrying the variant of interest with a MFS model, which should significantly alter the cardiovascular phenotype. Depending on the function and evolutionary conservation of the identified modifier gene, zebrafish or mouse models will be used.

Alternatively, the identified modifier will be functionally validated using the cutting-edge CRISPR/Cas9 genome editing technology in the available and thoroughly functionally characterized iPSC-VSMC lines.

Further evidence for a modifying role of the most interesting candidate genes will be obtained by performing targeted re-sequencing of these genes' coding and regulatory sequences in, again, the 25% most and least severely cardiovascular affected MFS cases of a large replication cohort consisting of more than 3000 clinically and molecularly (FBN1 mutation-positive) characterized index cases.

Whenever possible, segregation of the remaining candidate modifier variants with protection from TAAD will be investigated in available gDNA samples of the probands' relatives carrying the FBN1 mutation.

Eligibility Criteria:
Inclusion Criteria:

* Participants with proven mutation (p.Ile2585Thr;c.7754C\>T) in the FBN1 gene

Exclusion Criteria:

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