GLOMERULAR FRAGILITY AND VASCULAR BARRIER INSTABILITY INDUCED BY A COMMON EAST ASIAN LAMININ β2 VARIANT: ROLE OF THE LF DOMAIN

Laminins are critical structural components of basement membranes, with their diverse isoforms conferring tissue-specific functions. Mutations in Laminin β2 gene (LAMB2) are known to cause Pierson syndrome, a severe congenital nephrotic syndrome often accompanied by ocular abnormalities. However, milder clinical presentations have been associated with variants, particularly those affecting the short arm domains.  While the major globular and LE domains have been extensively characterized, the precise biological role of the laminin short arm LF domain, which is part of the short arm domains, has remained elusive. We previously demonstrated that variants in LF domain enhances integrin binding in vitro (Kikkawa et al. JCI Insight 2021). Here, we report the first in vivo investigation of the LF domain using the mouse G702R substitution, which is the ortholog of the human G699R variant and is enriched in East Asian populations (allele frequency 0.008).

We generated a CRISPR-Cas9-mediated knock-in mouse model harboring the Lamb2 G702R substitution. Homozygous and heterozygous G702R mice were subjected to phenotypic analysis, including baseline proteinuria measurement and ultrastructural studies. Glomerular stress was induced by adriamycin administration to evaluate barrier stability. Mechanistic insights were sought through RNA sequencing of isolated glomeruli and immunostaining. Ocular examination was performed to investigate systemic vascular barrier integrity.

Unlike classic Lamb2 loss-of-function mutations, which cause early postnatal lethality and prevent long-term observation, homozygous and heterozygous G702R mice developed normally without early lethality or baseline proteinuria, allowing for in-depth, long-term functional analysis. However, ultrastructural studies unexpectedly revealed a prominent loss of glomerular endothelial fenestrae in mutant mice at baseline, suggesting a latent fragility of the glomerular filtration barrier. Upon adriamycin stress, both heterozygous and homozygous mutants exhibited significantly exacerbated proteinuria and mesangial expansion relative to wild-type controls, which was accompanied by greater effacement of podocyte foot processes and near-complete loss of endothelial fenestrations. Mechanistic insights from RNA sequencing and immunostaining implicated altered matrix signaling and mechanical stress in the glomeruli. Furthermore, ocular examination revealed abnormal corneal thickening and age-dependent retinal thinning, a phenotype suggestive of structural compromise of the blood-retinal barrier, which was not observed in wild-type mice.

This study conclusively demonstrates the in vivo pathogenicity of the common East Asian G699R allele and simultaneously　reveals a previously unrecognized function of the Laminin β2 LF domain in renal and ocular vascular barrier maintenance, specifically demonstrating the critical role of laminin in endothelial cell integrity. Our findings highlight the importance of evaluating ethnically enriched variants in genetic risk assessment and disease modeling, which has significant implications for personalized precision medicine.