A comprehensive approach for identifying filaggrin mutations and copy number variants by long-read sequencing

Abstract

Filaggrin (FLG) is essential for skin barrier function, but has highly diverse and complex mutations linked to various allergic and dermatological diseases. Current genotyping methods often fail to capture the full range of FLG variants, especially in regions with high sequence homology. To overcome this limitation, we developed a singleplex PCR method that amplifies FLG exon 3 using FLG-specific primers tailed with barcodes for sample identification, followed by long-read sequencing on the PacBio Sequel IIe system. After demultiplexing with the barcode sequences, pbmm2 and GATK HaplotypeCaller were used for alignment and variant calling, respectively. This method successfully identified single nucleotide variants, insertion-deletion variants, and copy number variations (CNVs), including several loss-of-function mutations. We also determined the FLG copy number in each allele, which ranged from 7 to 20 repeats. This comprehensive, convenient genotyping approach could significantly enhance diagnostic accuracy and personalized treatment strategies for allergy- and skin-related conditions.