Genome-wide Epistasis for Cardiovascular Severity in Marfan Study (GEMS)

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.

Study Overview

• Status: Recruiting
• Conditions: Marfan Syndrome; FBN1 Mutation
• Intervention / Treatment: Genetic: Saliva collection (screening of all participants); Genetic: Bloodsampling

Detailed Description

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.

• Study Type: Observational
• Enrollment (Estimated): 200

Contacts and Locations

• Study Contact: Name: Bart Loeys, Prof,MD,PhD; Phone Number: ++32-3-2759768; Email: bart.loeys@uantwerpen.be
• Study Contact Backup: Name: Paul Coucke, Prof,PhD,Ing; Phone Number: ++32-9-3323634; Email: paul.coucke@ugent.be

Study Locations

• Belgium
  • Prins Boudewijnlaan 43/6
    • Edegem, Prins Boudewijnlaan 43/6, Belgium, 2650
      • Recruiting
      • University Hospital Antwerp
      • Contact: Bart Loeys; Phone Number: +3232759768; Email: bart.loeys@uantwerpen.be
      • Contact: Paul Coucke; Phone Number: +3293323634; Email: paul.coucke@ugent.be
      • Sub-Investigator: Julie De Backer, Prof,MD,PhD
      • Sub-Investigator: Laura Muiño Mosquera, MD,PhD
      • Sub-Investigator: Josephina Meester, MD,PhD
      • Sub-Investigator: Aline Verstraeten, MD,PhD

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Child; Adult; Older Adult
• Accepts Healthy Volunteers: No

• Sampling Method: Probability Sample

Study Population

participants with Marfan syndrom

Description

Inclusion Criteria:

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

Exclusion Criteria:

-

Study Plan

How is the study designed?

Number of groups / cohorts

2

Cohorts and Interventions

Group / Cohort	Intervention / Treatment
AMC (Affected Mutation Carrier); FBN1 mutation (Marfan Syndrome) - Phenotype cardiovascular severe outcome	Genetic: Saliva collection (screening of all participants); All participants will give a salive sample (self-sampling kit) - selection of 25% extremes (UMC and AMC) participants for WGS and linkage analysis; Genetic: Bloodsampling; Based on the results of the WGS, a selection will be made of the 5% most extremes (UMC and AMC) participants for the iPSC-VSMC generation
UMC (Unaffected Mutation Carrier); FBN1 mutation (Marfan Syndrome) - Phenotype cardiovascular mild outcome	Genetic: Saliva collection (screening of all participants); All participants will give a salive sample (self-sampling kit) - selection of 25% extremes (UMC and AMC) participants for WGS and linkage analysis; Genetic: Bloodsampling; Based on the results of the WGS, a selection will be made of the 5% most extremes (UMC and AMC) participants for the iPSC-VSMC generation

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Molecular characterization of the assembled 25% extreme ends cohort (UMC and AMC).	Whole Genome Sequencing (WGS) and linkage analysis will be used for this purpose	September 2023

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Omics integration for modifier identification in the 5% extreme ends of the cohort (UMC and AMC)	The modifier genes will be identified by the generation of iPSC-VSMC's of MFS individuals from the 5% extreme ends of the spectrum.	September 2023
Functional validation of the modifiers.	For the validation of the modifiers the CRISP/Cas9 gene editing technology will be used.	2024
Replication of the identified modifiers in a large MFS cohort Time frame: December 2024	The evidence for a modifying role of the most interesting candidate genes will be obtained by performing targeted re-sequencing of these genes again in the 25% most and least severely cardiovascular affected MFS cases of a large replication cohort consisting of more than 3000 clinically and molecularly characterized index participants.	2024

Collaborators and Investigators

• Sponsor: University Hospital, Antwerp
• Collaborators: University Hospital, Ghent
• Investigators: Principal Investigator: Bart Loeys, Prof,MD,PhD, University Hospital, Antwerp; Study Chair: Paul Coucke, Prof,MD,Ing, University Hospital, Ghent

Publications and helpful links

General Publications

• Loeys BL, Dietz HC, Braverman AC, Callewaert BL, De Backer J, Devereux RB, Hilhorst-Hofstee Y, Jondeau G, Faivre L, Milewicz DM, Pyeritz RE, Sponseller PD, Wordsworth P, De Paepe AM. The revised Ghent nosology for the Marfan syndrome. J Med Genet. 2010 Jul;47(7):476-85. doi: 10.1136/jmg.2009.072785.
• Braverman AC. Medical management of thoracic aortic aneurysm disease. J Thorac Cardiovasc Surg. 2013 Mar;145(3 Suppl):S2-6. doi: 10.1016/j.jtcvs.2012.11.062. Epub 2012 Dec 20.
• Verstraeten A, Luyckx I, Loeys B. Aetiology and management of hereditary aortopathy. Nat Rev Cardiol. 2017 Apr;14(4):197-208. doi: 10.1038/nrcardio.2016.211. Epub 2017 Jan 19.
• von Kodolitsch Y, De Backer J, Schuler H, Bannas P, Behzadi C, Bernhardt AM, Hillebrand M, Fuisting B, Sheikhzadeh S, Rybczynski M, Kolbel T, Puschel K, Blankenberg S, Robinson PN. Perspectives on the revised Ghent criteria for the diagnosis of Marfan syndrome. Appl Clin Genet. 2015 Jun 16;8:137-55. doi: 10.2147/TACG.S60472. eCollection 2015.
• Groth KA, Gaustadnes M, Thorsen K, Ostergaard JR, Jensen UB, Gravholt CH, Andersen NH. Difficulties in diagnosing Marfan syndrome using current FBN1 databases. Genet Med. 2016 Jan;18(1):98-102. doi: 10.1038/gim.2015.32. Epub 2015 Mar 26.
• De Backer J, Loeys B, Leroy B, Coucke P, Dietz H, De Paepe A. Utility of molecular analyses in the exploration of extreme intrafamilial variability in the Marfan syndrome. Clin Genet. 2007 Sep;72(3):188-98. doi: 10.1111/j.1399-0004.2007.00845.x.
• Franken R, Teixido-Tura G, Brion M, Forteza A, Rodriguez-Palomares J, Gutierrez L, Garcia Dorado D, Pals G, Mulder BJ, Evangelista A. Relationship between fibrillin-1 genotype and severity of cardiovascular involvement in Marfan syndrome. Heart. 2017 Nov;103(22):1795-1799. doi: 10.1136/heartjnl-2016-310631. Epub 2017 May 3.
• Renard M, Muino-Mosquera L, Manalo EC, Tufa S, Carlson EJ, Keene DR, De Backer J, Sakai LY. Sex, pregnancy and aortic disease in Marfan syndrome. PLoS One. 2017 Jul 14;12(7):e0181166. doi: 10.1371/journal.pone.0181166. eCollection 2017. Erratum In: PLoS One. 2018 May 14;13(5):e0197631.
• Granata A, Serrano F, Bernard WG, McNamara M, Low L, Sastry P, Sinha S. An iPSC-derived vascular model of Marfan syndrome identifies key mediators of smooth muscle cell death. Nat Genet. 2017 Jan;49(1):97-109. doi: 10.1038/ng.3723. Epub 2016 Nov 28.

Study record dates

Study Major Dates

• Study Start (Actual): November 30, 2020
• Primary Completion (Estimated): October 31, 2025
• Study Completion (Estimated): October 31, 2025

Study Registration Dates

• First Submitted: February 5, 2024
• First Submitted That Met QC Criteria: February 5, 2024
• First Posted (Estimated): February 13, 2024

Study Record Updates

• Last Update Posted (Estimated): February 13, 2024
• Last Update Submitted That Met QC Criteria: February 5, 2024
• Last Verified: January 1, 2024

More Information

Terms related to this study

• Keywords: Rare disease; Thoracic aortic disease; Genetic modifiers; Congenital abnormalities; p.Ile2585Thr;c.7754T>C; Aortopathies; TAAD; Thoracic aortic rupture
• Additional Relevant MeSH Terms: Heart Diseases; Cardiovascular Diseases; Congenital Abnormalities; Genetic Diseases, Inborn; Musculoskeletal Diseases; Connective Tissue Diseases; Bone Diseases; Heart Defects, Congenital; Cardiovascular Abnormalities; Musculoskeletal Abnormalities; Abnormalities, Multiple; Bone Diseases, Developmental; Limb Deformities, Congenital; Marfan Syndrome; Arachnodactyly
• Other Study ID Numbers: 20/08/087; 1S81820N (Other Grant/Funding Number: FWO (Foundation for national research Belgium)); B3002021000292 (Other Identifier: Belgian number)

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No