VEDS Research: Zebrafish Model
Elucidating functional significance of ‘Variants of Uncertain Significance’ by generating and characterising a zebrafish model for Vascular Ehlers-Danlos Syndrome
Annabelle’s Challenge announces £50,000.00 grant for new scientific vEDS research project.
31st May, 2024
We're delighted to have reaffirmed our objective to enabling research in vascular EDS (vEDS) by awarding a £50,000.00 grant to a University of Sheffield research project ‘Elucidating functional significance of ‘Variants of Uncertain Significance’ by generating and characterising a zebrafish model for Vascular Ehlers-Danlos Syndrome.’
This project aims to investigate the genetic alterations found in patients with vascular Ehlers -Danlos syndrome (vEDS). These alterations are officially termed Variants of Uncertain Significance (VUS), as there is currently insufficient information regarding their likelihood of causing disease (pathogenic) or not.
Although the genetic heterogeneity present in Vascular Ehlers-Danlos Syndrome is well understood, the pathways linking genetic alterations and the various variants of uncertain significance to the disease phenotype remain obscure.
The primary objective is to identify and reclassify the VUS most prevalent in patients according to the criteria set forth by the Association for Clinical Genomic Science (ACGS) and the American College of Medical Genetics and Genomics (ACMG). VUS that remain significant will undergo functional studies using zebrafish, a small fish widely used in scientific research. Employing advanced techniques, the Balasubramanian group will edit the fish genome, generating a zebrafish model with mutations in the genes responsible for collagen production, like the patients with vEDS. Subsequently, they will analyse how close to vascular EDS this zebrafish model is so it provides a way into using zebrafish as a disease model to further clarify the significance of these VUS.
In addition to these functional studies, this animal model will also be suitable for drug screening and potentially for future gene therapy research.
The work is being overseen by Prof Meena Balasubramanian who is an academic clinical geneticist based in Sheffield. She has recently taken over the role of Clinical Director of Research at Sheffield Children’s Hospital.
Meena Balasubramanian, PI on this project said, “I am really grateful to Annabelle’s challenge for supporting this really exciting and worthwhile project on exploring VUS in vascular EDS which is a growing problem with advanced genetic testing and using zebrafish as a model to understand the underlying cause of vEDS.”
Jared Griffin, Founder & CEO “This is an exciting project and a turning point for the charity as we fund scientific research for vascular EDS. Variants of uncertain significance is a very important area of work and I am delighted this is now being researched through the University of Sheffield with Prof Balasubramanian leading the project.”
Why use Zebrafish?
The zebrafish (Danio rerio) is a model organism widely used in biological research. It is a fresh water fish, part of the Cyprinids family in the ray-finned fish class.
Zebrafish and human genomes share around 70% homology. Moreover, 82% of morbidity-associated genes in human have been linked to at least one ortholog in the zebrafish. These similarities support the relevance of the model, but the advantages of zebrafish are also practical.
First, the zebrafish is a small organism and requires less infrastructure and nursing than mammalian models such as rodents. Their generation time is longer, approximately three to four months compared to 1.5 months for mice, but the descendants are far more numerous, with hundreds of eggs by clutch spawn every two or three days.
Rob Reyner Research Funding
Funding for this research project is granted from a donation of £50,000.00 in memory of Rob Reyner.
Rob joined Annabelle's Challenge in May 2017 and attended the world’s first ever vascular EDS Conference held during the same month at The Village Hotel, Bury.
He was a keen advocate of our work to help others affected by vascular EDS despite his own suffering and the loss of his father and brother to the same condition.
Rob was one of our main supporters hosting his annual Eurovision party to raise awareness and funds for REDS4VEDS and Annabelle's Challenge.
He would have attended our second Conference on 14th May 2022, however it clashed with his Eurovision 2022 party, sadly he passed away the same day.
Rob along with his family and friends have donated a significant amount of funds for Annabelle’s Challenge.
His legacy continues ensuring Rob's wishes are fulfilled benefiting the wider vEDS community both now and in the future, globally.
Research Project Update
The initial proposal funded by Annabelle’s Challenge had the following aims:
- Develop mutant zebrafish model for vEDS using the CRISPR system
- Develop functional studies for vEDS zebrafish model validation
- Identify variants of uncertain significance (VUS) in COL3A1 causing vEDS through extraction of diagnostic laboratory data from the national reference lab data
- Generate a database of previously reported VUS and collect clinical data from this data extraction
- Reclassify the VUS using current ACMG criteria to enable updates from likely pathogenic variants (class 3 variants) to pathogenic or likely pathogenic variants (class 4 or 5 variants)
- Establish a list of ‘hot’ VUS that can be taken forward in future work through generation of Crips knick-in and compare with established vEDS zebrafish model
We have made many strides forward in our research in the Balasubramanian lab @ The University of Sheffield. This has involved the reclassification of several VUS, the generation of a stable zebrafish model and the generation and characterisation of a ‘F0 crispant’ zebrafish model.
This project has been worked on by Prof Meena Balasubramanian (Principal Investigator), Dr Daniel Baird (Postdoctoral Research Associate) and Nataliya Pidlisnyuk (MSc Intercalating Medical Student).
Reclassification of VUS
Reclassification of VUS is a complex, labour-intensive process. We have investigated VUS from patients with COL3A1 variants at the Sheffield Diagnostic Genetics Service (the UK National Reference Diagnostics Laboratory) and reclassified a number of variants.
Prior to reclassification, no variants were thought to be ‘likely pathogenic’ (likely to be a variant that causes vEDS), but our reclassification increased this to 15%, with 23% being reclassified as ‘likely benign’ (variant unlikely to cause vEDS). Furthermore, we have reclassified 2 variants that were previously VUS to pathogenic (variant causes vEDS). This essentially means in total nearly half of the patients had a revised/ confirmed diagnosis based on this project.
The rest of the 54% of variants remained as VUS, with 23% now being reclassified as a ‘hot VUS’, where we have evidence that suggests these variants may be disease causing but the laboratory strict evidence is not strong enough yet.
When we compared patients in our Sheffield cohort to those already reported in literature, we found that patients in the Sheffield cohort shared similar clinical features of vEDS with those already reported, aligning with expectations that many of these patients could become diagnosed with vEDS.
For the 54% of remaining VUS, we would now aim to generate a functional model in order to assist in the reclassification of them. A model which shows that these VUS have similar clinical signs to known vEDS patients would provide evidence to confirm these as disease-causing. Based on the shared genetics and protein structures of certain VUS from our Sheffield cohort, we aim to generate zebrafish models for 2 VUSs.
Generation of a Stable Zebrafish Model
To help us reclassify VUS in vEDS patients, we have been able to generate a stable zebrafish model for mutations in the zebrafish col5a1 gene. We are using col5a1 in zebrafish to model COL3A1 vEDS variants because zebrafish do not have type III collagen present within their anatomy. However, other research studies have shown that type V collagen can be used as a surrogate for type III collagen.
To this end, we have used CRISPR-Cas9 genetic editing techniques to cause a mutation in the zebrafish col5a1 gene in order to ‘knockout’ this gene, meaning that the gene will not function. We have identified specific zebrafish adults which have a mutation in col5a1, and have raised two generations of zebrafish to adulthood with mutations in the col5a1 gene (which takes between 3-4 months per generation).
As we have generated two generations of ‘mutant’ fish, we can call this a ‘stable line’, as only the mutation we want will be present within these fish. We have generated two stable lines for mutations in the col5a1 gene - one with a 4 base pair deletion and one with a 2 base pair deletion. These stable lines will allow us to model the impact of vEDS when there are mutations where the gene does not function at all.
