A new integrated genetic and transcriptomic approach for investigating DUX4 and DUX4C

The double homeobox 4 gene (DUX4) and its centromeric paralogue, DUX4C, reside in the subtelomeric region of chromosome 4 and have been implicated in facioscapulohumeral muscular dystrophy (FSHD) and cancers. However, the high sequence similarity between these genes, together with the widespread presence of DUX4-like paralogues across the human genome, has hindered accurate genotyping and expression profiling using short-read sequencing. To elucidate the genetic architecture and potential disease-associated functions of DUX4 and DUX4C, we first identified two distinct DUX4C haplotypes with expression quantitative trait effects-DUX4C-4qα and DUX4C-4qβ. We then integrated them with known DUX4 haplotypes to generate a reference genome, D4Ref-T2T, using long-read sequencing. Haplotype analysis indicated strong linkage disequilibrium between DUX4C and DUX4 haplotypes (r² = 0.86). We further characterized full-length DUX4C mRNA isoforms and established a corresponding transcriptome reference. Applying these resources to breast tumor, FSHD, and lymphoblastoid cell line datasets revealed that DUX4 is expressed in both breast tumor and FSHD tissues, whereas DUX4C shows expression only in breast tumors. Differential gene expression and Gene Ontology enrichment analyses further suggested that DUX4C expression is associated with activation of pathways involved in leukocyte differentiation, chemotaxis, and cell migration.