Abstract
COL4A1- and COL4A2-related disorders cause a broad spectrum of problems comprising abnormal
brain development, brain hemorrhage at any age, aneurysms (local dilatations) of the brain arteries,
but also eye or renal problems. In addition, COL4A1 was recently identified as a genetic modifier in Marfan syndrome. We studied the presence of COL4A1 and COL4A2 variants in two patient cohorts; a cerebral palsy (CP) and a TAA (thoracic aortic aneurysm) cohort. This led to a specific interest in the COL4A2 NC1 domain. A burden analysis demonstrated a statistically significant overrepresentation of COL4A2 NC1 variants in the CP cohort. Furthermore, we identified the NC1 variant p.Arg1662His in 3 TAA patients and 3 CP patients of Moroccan descent. In 5 cases in combination with the helical variant p.Met1355Thr. The latter is suggestive of a shared "risk haplotype". The p.Arg1662His variant was significantly overrepresented in Moroccan patients in our cohorts compared to a Moroccan control cohort. In addition,
We will study the cellular effects of NC1 variants using patient fibroblasts in order to assess (1) the levels of endoplasmatic reticulum stress and activation of the unfolded protein response and (2) alterations in Akt-FAK-mTOR signaling and procaspase 8 and 9 expression. Fibroblasts were collected from patients harboring (1) the COL4A2 variant p.Arg1662His, (2) the COL4A2 variant p.Arg1662His in combination with the COL4A2 variant p.Met1355Thr and (3) the pathogenic COL4A2 p.Gly1353Ala as a positive control. Three wild-type fibroblasts are used as negative controls.
Secondly, we will develop a zebrafish model to study the effect of COL4A2 NC1 variants.
We will start with the introduction of the pathogenic COL4A2 p.Gly1353Ala variant and study the effect on zebrafish development using a fish that has fluorescent blood vessels in order to easily pick up abnormal vessels. We will study the occurrence of brain haemorrhage, changes in movement patterns and the basement membrane, a structure that stabilizes the wall of blood vessels and measure the aortic diameter. When a reliable read-out is identified, we will introduce NC1 variants in the zebrafish model to assess their effect.
This project is the first study to investigate the contibution of specific COL4A2 NC1-domain variants in pathology. When our findings are corroborated by functional studies, it would also be the first identification of a population-specific COL4A2-related risk haplotype associated with cerebral and aortic vascular pathology, which is an important finding in the age of personalized medicine.
Another novelty is the development and use of a zebrafish model to study functional effects of COL4A2 variants using (CRISPR)/Cas9 technology. The model would enable not only functional analysis of additional variants of unknown significance in cerebrovascular pathology and TAA, but additionally allows studies regarding the pathogenic mechanisms underlying different types of COL4A2-mutations. This will help in identifying potential therapeutic strategies. Eventually, the model is suited for testing of potential treatment strategies in vivo, enabling monitoring of the therapeutic effect, as well as unwanted side-effects.
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