SsCSFLs have a strong association with connective tissue diseases (CTDs) including Marfan syndrome and Loeys-Dietz syndrome. Patients with ssCSFLs without specific diagnoses often have nonspecific CTD manifestations, suggesting that mutations in extracellular matrix proteins underlie more common presentations of this condition.

To investigate whether patients with spontaneous spinal cerebrospinal fluid leaks (ssCSFLs) have a higher frequency of rare variants in a single gene that could predispose them to this condition, and to study these variants' effects on cell function and dural homeostasis in mice.

Our case-control study of 49 Type 1b ssCSFL probands and 3 control groups (n = 2244, 1428, 1826) identified FBN2 as a top candidate gene. Recombinant WT and mutant fibrillin-2 fragments were synthesized using the Expi293 system and tested for human dural fibroblast binding and integrin involvement in vitro. Fbn2 variants were introduced in C57BL/6 mice using CRISPR-Cas9, which underwent lumbar infusion testing to determine dural integrity and resistance to rupture, with Fbn1^C3041G/+ (Marfan syndrome model) as a positive control group. Single-nuclear RNA sequencing of mouse dura (n = 3-4 mice/group) was analyzed using Seurat in R.

We report a significantly increased burden of rare variants in FBN2 in patients with type 1b ssCSFLs. Two of these mutations reduce human dural fibroblast adhesion to fibrillin-2 fragments in vitro. One disrupts a known integrin binding motif in the TB4 domain; another affects a completely novel binding site in the TB7 domain. Mice harboring the fibrillin-2 TB4 variant (Fbn2^D1581V/+) and a mouse model of Marfan syndrome (Fbn1^C1041G/+) demonstrate a pronounced predisposition for dural rupture upon controlled leak induction. Single-nuclear RNA sequencing of Fbn2^D1581V/+ and Fbn1^C1041G/+ mouse dura demonstrates dysregulation of tropoelastin expression in dural fibroblasts.

These data suggest mutations in FBN2 that alter cellular adhesion and/or the synthetic repertoire of matrix elements may lead to increased susceptibility to ssCSFLs.