TNXB Related Hypermobility

Abstract

Hypermobile Ehlers-Danlos Syndrome (hEDS) is a heritable connective tissuedisorder, characterized by joint hypermobility, musculoskeletal abnormalities, abnormal skin, and chronic pain. A clear genetic cause for hEDS remains unidentified, making diagnosis reliant on clinical examination by a trained healthcare provider. As a result, many individuals experience delays in diagnosis, typically greater than a decade, ultimately delaying interventions that could mitigate hEDS-related disability. This gap has prompted research efforts aimed at identifying genetic markers or biomarkers that could make the diagnostic process more objective and efficient, while also providing potential therapeutic targets. In the presented studies, we explore genetic variation in TNXB, a gene previously implicated in EDS, in a series of individuals and families affected by hEDS. Additionally, we investigate expression differences using skin tissue samples and single-cell RNA sequencing (scRNA-seq). Pathogenic variants in both alleles of TNXB, which codes for the protein Tenascin-X,are responsible for classic-like EDS, one of the 13 recognized types of EDS. Heterozygous mutations in TNXB are thought to contribute to the development of hEDS, which is the most common subtype, accounting for 90% of EDS cases. However, research into TNXB variants in hEDS is limited by the technical challenges posed by TNXA, a closely related pseudogene. Due to the high sequence similarity between TNXB and TNXA, short-read sequencing, which is the standard for clinical genetic testing, cannot reliably distinguish between the two genes, leading to potential misalignment and the formation of a dysfunctional TNXB/A chimera. To address this, we employed long-read sequencing and quantitative polymerase chain reaction (qPCR) to study TNXB variants in regions confounded by TNXA. This approach is the first investigation to our knowledge of TNXB variants in hEDS using long-read sequencing technologies. Our analysis did not identify any pathogenic variants in TNXB among individualswith hEDS. Additionally, there was no significant difference in the frequency of rare or common missense variants between the hEDS series and unaffected controls. While TNXB mutations may still play a role in a rare subtype of EDS, a larger series of individuals with hEDS be required to establish an accurate variant prevalence. The Hypermobile Ehlers- Danlos Genetic Evaluation (HEDGE) study has collected DNA from more than 1000 individuals with hEDS and can serve as an expanded series. In addition to genetic analysis, we used scRNA-seq to investigate the cellular andmolecular features of skin tissue from five healthy controls and five individuals with hEDS. scRNA-seq is a powerful tool for examining cellular heterogeneity within tissues, providing insights into disease mechanisms at the single-cell level. Previous studies of hEDS fibroblasts have identified an increased frequency of myofibroblasts, which are characterized by the dysregulation of the ACTA2 gene (encoding smooth muscle α-actin, α-SMA). Myofibroblasts are specialized fibroblasts that differentiate in response to inflammation and contribute to fibrosis, suggesting that inflammation may play a significant role in the complex hEDS phenotype. In our analysis, while we did not observe an increase in myofibroblasts, weidentified a notable increase in APOE and CCL19 pro-inflammatory fibroblasts, as well as an expansion of immune cell populations, including T-cells, macrophages, and dendritic cells. T-cells are essential in regulating inflammation and are commonly elevated in autoinflammatory conditions. Given that T-cells are also an established therapeutic target for modulating inflammation, these findings suggest a potential target for future therapeutic interventions aimed at managing inflammation in hEDS. These studies represent an important step in advancing our understanding of thegenetic and cellular underpinnings of hEDS. While no direct pathogenic variants in TNXB were identified using long-read sequencing, the insights gained from this work will inform future research and diagnostic approaches. The identification of immune-related pathways, particularly the role of T-cells in hEDS, opens new avenues for therapeutic development aimed at addressing the chronic inflammation seen in this disorder. Further studies, particularly those utilizing larger series and advanced sequencing techniques, are needed to fully elucidate the complex genetic and immunological mechanisms contributing to hEDS.