RNA-Seq of Cultured Peripheral Blood Lymphocytes Improves Identification of Cryptic Splicing Defects in Rare Disease Diagnostics

  • Hum Mutat. 2026 Jan 8:2026:9635551. doi: 10.1155/humu/9635551.
Jinlin Ren  1  2 ,  Congling Dai  1  2  3 ,  Fei Meng  2 ,  Pan Zhang  1  4 ,  Chunbo Xie  3 ,  Wenjuan Xiao  1 ,  Wenbin He  1  2 ,  Shimin Yuan  2 ,  Xiurong Li  2 ,  Qianjun Zhang  2  3 ,  Weiling Tang  2 ,  Liang Hu  2  3 ,  Zixu Chen  3 ,  Guangxiu Lu  1  2  3  4  5 ,  Juan Du  3  4 ,  Sicong Zeng  1  2  4  5 ,  Ge Lin  2  3  4  5
Affiliations
  • 1. Hunan Guangxiu Hospital, Hunan Guangxiu Hi-tech Life Technology Co., Ltd, Hunan Normal University, Changsha, Hunan, China, hunnu.edu.cn.
  • 2. Clinical Research Center for Reproduction and Genetics in Hunan Province, Reproductive and Genetic Hospital of Citic-Xiangya, Changsha, Hunan, China, zxxyyy.cn.
  • 3. Institute of Reproduction and Stem Cell Engineering, School of Basic Medical Science, Central South University, Changsha, China, csu.edu.cn.
  • 4. National Engineering and Research Center of Human Stem Cells, Changsha, China.
  • 5. Hunan International Scientific and Technological Cooperation Base of Development and carcinogenesis, Changsha, Hunan, China.
PMID: 41523913 DOI: 10.1155/humu/9635551
Abstract

Accurate identification of the genetic determinants of rare diseases is essential for effective recurrence-risk management and informed reproductive decision-making. Although whole-exome Sequencing (WES) and whole-genome Sequencing (WGS) have significantly improved diagnostic capabilities, a subset of affected families still receives no definitive molecular diagnosis. RNA Sequencing (RNA-seq) has emerged as a promising complementary diagnostic tool, yet its clinical implementation in the context of preconception genetic counseling remains underexplored. We used phytohemagglutinin-activated peripheral blood cells (PHACs) as a robust RNA source and enhanced conventional RNA-seq through the integration of three analytical innovations: (1) transcript isoform distribution (TID) analysis, (2) realignment against the MANE (Matched Annotation from NCBI and EMBL-EBI) reference transcriptome, and (3) pharmacological induction-based cryptic splicing detection. This optimized pipeline was applied to 55 rare-disease families with negative WES/WGS results who were undergoing preconception genetic counseling. Based on prior evaluations, families were grouped as VUS (n = 7), suspected-gene/variant-negative (n = 10), and unsolved/no-candidate (n = 38). PHACs showed reduced interindividual variability and higher RNA integrity than fresh PBMCs (median RIN: 9.77 vs. 8.97; p < 0.0001). The optimized workflow improved diagnostic yield by 2.2-fold (20% vs. 9%). Stratified analysis revealed positive rates of 71% (VUS), 40% (suspected-gene/variant-negative), and 5.2% (unsolved/no-candidate). Among the 11 positive cases, 10 received definitive diagnoses, leading to diverse reproductive decisions. This enhanced RNA-seq workflow provides a clinically applicable and scalable strategy for improving molecular diagnostics in reproductive and preconception settings, offering a valuable model for future clinical transcriptomics.

Keywords
RNA sequencing (RNA-seq); peripheral blood; phytohemagglutinin-activated peripheral blood cells (PHACs); preconception genetic counseling; rare diseases.
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