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

Helicase-inactivating BRIP1 mutation yields Fanconi anemia with microcephaly and other congenital abnormalities

Fanconi anemia is a genetically and phenotypically heterogeneous disorder characterized by congenital anomalies, bone marrow failure, cancer, and sensitivity of chromosomes to DNA cross-linking agents. One of the 22 genes responsible for Fanconi anemia is BRIP1, in which biallelic truncating mutations lead to Fanconi anemia group J and monoallelic truncating mutations predispose to certain cancers. However, of the more than 1000 reported missense mutations in BRIP1, very few have been functionally characterized. We evaluated the functional consequence of BRIP1 p.R848H (c.2543G > A), which was homozygous in two cousins with low birth weight, microcephaly, upper limb abnormalities, and imperforate anus and for whom chromosome breakage analysis of patient cells revealed increased mitomycin C sensitivity. BRIP1 p.R848H alters a highly conserved residue in the catalytic DNA helicase domain. We show that BRIP1 p.R848H leads to a defect in helicase activity. Heterozygosity at this missense has been reported in multiple cancer patients but, in the absence of functional studies, classified as of unknown significance. Our results support that this mutation is pathogenic for Fanconi anemia in homozygotes and for increased cancer susceptibility in heterozygous carriers

Genomic analysis of inherited hearing loss in the Palestinian population

The genetic characterization of a common phenotype for an entire population reveals both the genetic epidemiology of that phenotype and the power of family-based, population-wide genomic analysis. We characterized the genetics of hearing loss throughout the Palestinian population of the West Bank and Gaza. In families with no prior history of hearing loss, we estimate that 56% of hearing loss is genetic and 44% is not genetic. For most families with inherited hearing loss, causal genes and mutations were identified. Most inherited hearing loss in the population was attributable to consanguinity. Given the ongoing decline in consanguineous marriage, inherited hearing loss will likely be much rarer in the next generation