Long-read full-length transcriptome sequencing reveals multilayered sex-associated isoform regulation in thyroid carcinoma

Abstract Background Sex disparities in thyroid carcinoma are well recognized, yet their molecular basis at the transcript isoform level remains poorly understood. We aimed to determine whether sex-associated molecular differences extend beyond gene-level expression to coordinated remodeling of transcript isoforms. Methods Tumor tissues from ten thyroid carcinoma patients (four males, six females) were profiled by Oxford Nanopore Technologies (ONT) full-length transcriptome sequencing. Gene-level, transcript-level and post-transcriptional analyses, including differential expression, alternative splicing, alternative polyadenylation and differential transcript usage, were integrated. External validation was performed by exon-usage analysis of the TCGA-THCA cohort (505 primary tumors). Results We identified 14,420 genes and 32,269 transcripts, including 1,064 novel genes and 9,755 novel transcripts. Transcript-level analysis detected 2,052 differentially expressed transcripts, nearly twice the number of differentially expressed genes (1,144), with 627 alterations detectable only at the isoform level. Sex-associated analysis revealed 68 differential splicing events (predominantly exon skipping), 340 alternative polyadenylation events, 94 3′UTR remodeling events and 956 transcripts with altered isoform preference. Integration across layers yielded 56 consistently dysregulated candidates, represented by SNHG5 and PTCSC2. External TCGA-THCA exon-usage analysis reproduced sex-associated signal at JPX and SNHG5 that was undetectable at the gene-expression level. Using GTEx normal thyroid (v8, n = 578; v10, n = 587) and TCGA adjacent-normal tissue (n = 59) as references, only 28 genes (26 in v10) showed sex-biased expression in normal thyroid, almost all sex-chromosomal or immunoglobulin genes, and no autosomal gene passed correction in tumours; 1,917 of the 2,139 sex-associated event genes (89.6%) showed no sex-biased gene-level expression in either normal thyroid or tumour. Conclusions Sex-associated regulation in thyroid carcinoma is mediated through coordinated changes in transcript abundance and transcript identity, establishing isoform-resolved profiling as an essential complement to conventional gene-level analysis of endocrine tumor heterogeneity.