Identification of modifier genes underlying intra-familial phenotypic variability in zebrafish OI models using whole exome sequencing and linkage analysis

Clinical variability in OI patients carrying an identical causal variant is frequently observed. This suggests that modifier genes contribute to the phenotypic severity through a network of interactions with the causative gene. Zebrafish is known to be a powerful model to study skeletal disorders . They are highly suitable to study intra-familial variability because of a high level of genomic variation, similarity to human, and because high numbers of progeny with the same causal mutation can be obtained. Therefore, the main objective of this study was to identify modifier genes underlying the phenotypic variability observed in existing OI zebrafish models. We studied a mutant zebrafish line carrying a glycine substitution in the col1a2 gene. Deep skeletal phenotyping was done using a combination of X-ray and Alizarin red mineral staining. Exome sequencing of tail fin DNA was performed on a NovaSeq 6000 illumina sequencer, followed by SNP-based linkage analysis using Superlink Online SNP tool. Deep phenotyping in a large number of col1a2 mutants (n=18) obtained from a single set of parents, revealed a wide phenotypic variability in the vertebral column with variable mineralization, fracture incidence, scoliosis, and other skeletal abnormalities. Exome sequencing of the 6 most mildly and 6 most severely affected col1a2 mutants followed by SNP-based linkage analysis, revealed a potentially linked region on chromosome 14 which segregates with the phenotypic severity in the col1a2 OI model. Haplotype analysis revealed that this genomic region is 3.06 Mb in size and contains 45 protein coding genes. We are currently validating and narrowing down this candidate region in order to identify potential modifier(s). We also aim to apply this strategy in other OI zebrafish models with a variable skeletal phenotype such as the fkbp10a KO zebrafish model. We showed that zebrafish is a promising model for the analysis of modifier genes involved in skeletal diseases, and most likely also in other disorders. Modifier genes represent promising targets for intervening in disease initiation and progression.