888 FLG allelic and mutation phasing with nanopore sequencing to study phenotypic variability in ichthyosis vulgaris

Loss-of-function (LoF) mutations in FLG are associated with ichthyosis vulgaris (IV) and atopic dermatitis. Patients with multiple LoF mutations tend to have more severe disease but a subset of compound heterozygous patients still display mild phenotypes despite a prediction of total loss of filaggrin expression. The underlying genetic mechanism of this phenotypic variability is unclear and might be explained by LoF mutations being located in cis, thus preventing the total loss of protein expression. To investigate this at the genetic level we used Oxford Nanopore sequencing to phase the entire coding region of FLG into parental alleles in 22 subjects with known genotypes and IV disease severity. This enabled detection and accurate phasing of LoF mutation patterns and allele-specific attributes such as FLG intragenic copy-number variation (CNV) into parental alleles for all samples. We also piloted adaptive sampling for FLG from total DNA to remove the necessity for amplicons and we achieved identical genetic concordance for LoF and CNV, with the addition of methylation status. Ultimately, we observed the absence of mutations in cis, thus hinting at additional factors responsible for IV phenotypic variability in compound heterozygotes while also revealing additional insight into FLG allelic structure. These results highlight the utility of long-reads for interrogating FLG and other repetitive genes for accurate genotyping and clinical diagnostics.