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Background: The filaggrin (FLG) protein, encoded by the FLG gene, is an intermediate filament-associated protein that plays a crucial role in the terminal stages of human epidermal differentiation. Loss-of-function mutations in the FLG exon 3 have been associated with skin diseases. The identification of causative mutations is challenging, due to the high sequence homology within its exon 3 (12,753 bp), which includes 10 to 12 filaggrin tandem repeats. With this study we aimed to obtain the whole FLG exon 3 sequence through PacBio technology, once 13-kb amplicons have been generated. Methods and results: For the preparation of SMRTbell libraries to be sequenced using PacBio technology, we focused on optimizing a 2-step long-range PCR protocol to generate 13-kb amplicons covering the whole FLG exon 3 sequence. The performance of three long-range DNA polymerases was assessed in an attempt to improve the PCR conditions required for the enzymes to function properly. We focused on optimization of the input template DNA concentration and thermocycling parameters to correctly amplify the entire FLG exon 3 sequence, minimizing non-specific amplification. Conclusions: Taken together, our findings suggested that the PrimeSTAR protocol is suitable for producing the amplicons of the 13-kb FLG whole exon 3 to prepare SMRTbell libraries. We suggest that sequencing the generated amplicons may be useful for identifying LoF variants that are causative of the patients' disorders. [ABSTRACT FROM AUTHOR]

Background: Recent genome-wide studies revealed the molecular subtypes and mutational landscape of bladder cancer, which is the 10th most common cancer causing many deaths. ELF3 is one of the frequently mutated genes in bladder cancer with 14% alteration rate. It mainly functions as an epithelial transcription factor and its proper function is critical for the urothelium development. However, the impact of ELF3 mutations in bladder cancer is currently unknown. Methods and results: In this study, we analysed the gene expression data available for primary bladder cancer and bladder cancer cell lines according to the mutation status of ELF3. Our results show that de-regulated genes common in cell lines and primary tissue are primarily involved in ameboidal type cell migration and cell–cell junction organization. Additionally, we identify that ELF3-mutant cases in primary samples significantly overexpress PIK3C2B and ELF3 and PIK3C2B and ELF3 are significantly co-mutated in many cancer types. Our integrative analysis with existing Hi-C data further revealed the genes proximally located to ELF3, including PIK3C2B to be upregulated in ELF3 mutant cases, potentially as a result of truncated ELF3 protein product and subsequent changes in regulatory interactions. Conclusions: Our results provide important insights about how ELF3 mutation contributes to bladder tumorigenesis and uncover previously unknown dependencies. [ABSTRACT FROM AUTHOR]