Deciphering the pathogenesis of the musculoskeletal manifestations of Marfan Syndrome

Thesis

Marfan Syndrome, an autosomal dominant condition usually caused by FBN1 mutations, results in cardiovascular, ocular and musculoskeletal manifestations. Musculoskeletal features, including tall stature, kyphoscoliosis, and thoracic abnormalities, represent a significant burden to patients but are often overlooked. Studies suggest that TGF-β is an important pathogenic mediator in Marfan Syndrome. Prophylactic treatment with losartan, which is known to suppress TGFβ expression, has been shown to modify cardiovascular manifestations in murine and human Marfan Syndrome. My project characterised the musculoskeletal manifestations of the Fbn1C1039G/+ Marfan Syndrome mouse and explored the underlying molecular basis by assessing the growth plate phenotype using histology, bulk and single nuclear RNA sequencing. I also explored the effect of losartan on the skeletal phenotype.

MicroCT performed at 4, 6, 9, 12 and 26 weeks of age (n=3-22/group) showed that, across all ages, Fbn1C1039G/+ mice had increased lateral spine, femur and tibia lengths compared to controls, and more severe kyphosis, indicated by a reduced kyphosis angle. Histomorphometry at 6 weeks revealed increased tibial growth plate length and cellularity in Fbn1C1039G/+ mice, which normalised by 9 weeks. Continuous losartan treatment from 4-9 weeks (0.6g/l) reversed the skeletal phenotype, suggesting TGF-β pathway involvement in Marfan-related skeletal overgrowth.

To further elucidate molecular pathways underlying these changes, a novel method was developed to remove the tibial growth plates of control and treated, wildtype and Fbn1C1039G/+ 6 week old male mice. Bulk mRNA sequencing was performed on the isolated RNA, showing limited genotype effects, but a response to losartan 6 treatment. Subsequently, a novel nuclear isolation method was developed for single nuclear RNA sequencing from growth plate tissue. Sequencing RNA from single nuclei was successful and the data are soon to be analysed.

These findings confirm distinct skeletal and growth plate abnormalities in Fbn1C1039G/+ mice. Losartan's ability to attenuate these changes suggests a potential therapeutic strategy to arrest skeletal overgrowth in Marfan Syndrome patients. Furthermore, novel growth plate bulk tissue and nuclear isolation methods potentially offer significant methodological developments to support investigations into molecular mechanisms of skeletal abnormalities in Marfan Syndrome and other growth plate diseases.

Authors: Ślęczka, U

• DOI: 10.5287/ora-o86av11db
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: Bulk mRNA sequencing of the growth plate; snRNA sequencing of the growth plate
• Subjects: Histology; Marfan syndrome; Connective tissues; Microcomputed tomography; Transforming growth factors-beta; Growth plate; High-throughput nucleotide sequencing; Medicine; Musculoskeletal system--Abnormalities; Connective tissues--Diseases