Drosophila Col4a1 Glycine mutations highlight allelic heterogeneity and mechanistic pleiotropy.

Collagen IV (Col4) is a heterotrimer containing a triple helical domain broken up by short interruptions. Mutation of Glycine residues within the Glycine-X-Y triple helical repeat leads to genetically dominant disease in humans that affects multiple organ systems. Mouse and cell culture-based models have revealed allelic heterogeneity, resulting in a range of Col4 secretion defects depending on the position of the mutation. However, genetic background also affects phenotypic severity, making it challenging to understand the precise underlying molecular mechanisms driving disease. Here, we characterize an allelic series of dominant temperature-sensitive Drosophila Glycine mutations to identify the potential molecular mechanisms driving phenotypic heterogeneity. Analysis of developmental viability at the non-permissive temperature revealed that mutations show a range of developmental lethality that is not correlated with their position within the triple helix nor with the degree of Col4 secretion defect. Backcrossing the series of fly lines led to increased lethality for almost all alleles, highlighting the presence of genetic modifiers, which paradoxically led to a reduction in secretion defects; this further suggests that defective secretion cannot explain the allelic heterogeneity in mutant viability. Analysis of the Col4 network surrounding the central nervous system (CNS) revealed that Glycine mutations can also affect basement membrane (BM) structure and alter its mechanical properties. Additionally, fluorescent tagging of a Glycine mutant variant showed that the mutated trimer is sufficiently secreted and can be incorporated into the network to dominantly affect BM organization. These data reveal that Col4 Glycine mutations can cause both secretion and direct BM network defects, suggesting that Col4-related pathologies may be mechanistically pleiotropic.