massively-parallel-splicing-assay-to-examine-splicing-errors-caused

Massively Parallel Splicing Assay to Examine Splicing Errors Caused by Disease-Related Intronic Variants

Splicing errors represent 10-30% of the pathogenic mutations responsible for rare genetic disorders. RNA splicing ensures proper gene expression by ...

Academia Sinica

The study aims are to evaluate the associations between nasopharyngeal carcinoma (NPC) risk and cigarette smoking and to explore the effects of cigarette smoking on Epstein-Barr virus (EBV) infection for NPC risk.

Cigarette smoking increases the risk of nasopharyngeal carcinoma through the elevated level of IgA antibody against Epstein-Barr virus capsid antigen: A mediation analysis.

Both follicle stimulating hormone (FSH) and anti-mullerian hormone (AMH) are widely used to assess the ovarian reserve in women for in vitro fertilization (IVF). However, studies also showed that both AMH and FSH are significantly associated with age: as age increases, AMH decreases and FSH increases. This study aims to investigate the mechanism of age effect on IVF live birth rate, particularly through mediation and interaction by AMH and FSH.

Age effect on in vitro fertilization pregnancy mediated by anti-Mullerian hormone (AMH) and modified by follicle stimulating hormone (FSH).

For women undergoing in vitro fertilization (IVF), the clinical benefit of embryo transfer at the blastocyst stage (Day 5) versus cleavage stage (Day 3) remains controversial. The purpose of this study is to compare the implantation rate, clinical pregnancy rate and odds of live birth of Day 3 and Day 5 embryo transfer, and more importantly, to address the issue that patients were chosen to receive either transfer protocol due to their underlying clinical characteristics, i.e., confounding by indication.

Estimating the causal effect of embryo transfer day on clinical in vitro fertilization outcomes using propensity score matching.

Both entecavir (ETV) and tenofovir disoproxil fumarate (TDF) are first-line therapies for chronic hepatitis B (CHB), but their comparative effectiveness with regards to hepatitis B surface antigen (HBsAg) seroclearance remains unclear.

Effectiveness of entecavir vs tenofovir disoproxil fumarate for functional cure of chronic hepatitis B in an international cohort.

It is estimated that 10%-30% of disease-associated genetic variants affect splicing. Splicing variants may generate deleteriously altered gene product and are potential therapeutic targets. However, systematic diagnosis or prediction of splicing variants is yet to be established, especially for the near-exon intronic splice region. The major challenge lies in the redundant and ill-defined branch sites and other splicing motifs therein. Here, we carried out unbiased massively parallel splicing assays on 5,307 disease-associated variants that overlapped with branch sites and collected 5,884 variants across the 5' splice region. We found that strong splice sites and exonic features preserve splicing from intronic sequence variation. Whereas the splice-altering mechanism of the 3' intronic variants is complex, that of the 5' is mainly splice-site destruction. Statistical learning combined with these molecular features allows precise prediction of altered splicing from an intronic variant. This statistical model provides the identity and ranking of biological features that determine splicing, which serves as transferable knowledge and out-performs the benchmarking predictive tool. Moreover, we demonstrated that intronic splicing variants may associate with disease risks in the human population. Our study elucidates the mechanism of splicing response of intronic variants, which classify disease-associated splicing variants for the promise of precision medicine.

Mechanism and modeling of human disease-associated near-exon intronic variants that perturb RNA splicing.

Extremely early age at menarche, also called precocious puberty, has been associated with various cardiometabolic traits, but their shared heritability remains unclear.

Hypertension as a Novel Link for Shared Heritability in Age at Menarche and Cardiometabolic Traits.

Immunosuppression increases the risk of nosocomial infection in patients with chronic critical illness. This exploratory study aimed to determine the immunometabolic signature associated with nosocomial infection during chronic critical illness. We prospectively recruited patients who were admitted to the respiratory care center and who had received mechanical ventilator support for more than 10 days in the intensive care unit. The study subjects were followed for the occurrence of nosocomial infection until 6 weeks after admission, hospital discharge, or death. The cytokine levels in the plasma samples were measured. Single-cell immunometabolic regulome profiling by mass cytometry, which analyzed 16 metabolic regulators in 21 immune subsets, was performed to identify immunometabolic features associated with the risk of nosocomial infection. During the study period, 37 patients were enrolled, and 16 patients (43.2%) developed nosocomial infection. Unsupervised immunologic clustering using multidimensional scaling and logistic regression analyses revealed that expression of nuclear respiratory factor 1 (NRF1) and carnitine palmitoyltransferase 1a (CPT1a), key regulators of mitochondrial biogenesis and fatty acid transport, respectively, in natural killer (NK) cells was significantly associated with nosocomial infection. Downregulated NRF1 and upregulated CPT1a were found in all subsets of NK cells from patients who developed a nosocomial infection. The risk of nosocomial infection is significantly correlated with the predictive score developed by selecting NK cell-specific features using an elastic net algorithm. Findings were further examined in an independent cohort of COVID-19-infected patients, and the results confirm that COVID-19-related mortality is significantly associated with mitochondria biogenesis and fatty acid oxidation pathways in NK cells. In conclusion, this study uncovers that NK cell-specific immunometabolic features are significantly associated with the occurrence and fatal outcomes of infection in critically ill population, and provides mechanistic insights into NK cell-specific immunity against microbial invasion in critical illness.

Immunometabolic features of natural killer cells are associated with infection outcomes in critical illness.

Splicing variants are a major class of pathogenic mutations, with their severity equivalent to nonsense mutations. However, redundant and degenerate splicing signals hinder functional assessments of sequence variations within introns, particularly at branch sites. We have established a massively parallel splicing assay to assess the impact on splicing of 11,191 disease-relevant variants. Based on the experimental results, we then applied regression-based methods to identify factors determining splicing decisions and their respective weights.

SpliceAPP: an interactive web server to predict splicing errors arising from human mutations.

Untranslated regions (UTRs) contain crucial regulatory elements for RNA stability, translation and localization, so their integrity is indispensable for gene expression. Approximately 3.7% of genetic variants associated with diseases occur in UTRs, yet a comprehensive understanding of UTR variant functions remains limited due to inefficient experimental and computational assessment methods. To systematically evaluate the effects of UTR variants on RNA stability, we established a massively parallel reporter assay on 6555 UTR variants reported in human disease databases. We examined the RNA degradation patterns mediated by the UTR library in two cell lines, and then applied LASSO regression to model the influential regulators of RNA stability. We found that UA dinucleotides and UA-rich motifs are the most prominent destabilizing element. Gain of UA dinucleotide outlined mutant UTRs with reduced stability. Studies on endogenous transcripts indicate that high UA-dinucleotide ratios in UTRs promote RNA degradation. Conversely, elevated GC content and protein binding on UA dinucleotides protect high-UA RNA from degradation. Further analysis reveals polarized roles of UA-dinucleotide-binding proteins in RNA protection and degradation. Furthermore, the UA-dinucleotide ratio of both UTRs is a common characteristic of genes in innate immune response pathways, implying a coordinated stability regulation through UTRs at the transcriptomic level. We also demonstrate that stability-altering UTRs are associated with changes in biobank-based health indices, underscoring the importance of precise UTR regulation for wellness. Our study highlights the importance of RNA stability regulation through UTR primary sequences, paving the way for further exploration of their implications in gene networks and precision medicine.

Multiplexed assays of human disease-relevant mutations reveal UTR dinucleotide composition as a major determinant of RNA stability.

In eukaryotic cells, reactive oxygen species (ROS) serve as crucial signaling components. ROS are potentially toxic, so constant adjustments are needed to maintain cellular health. Here we describe a single-cell, mass cytometry-based method that we call signaling network under redox stress profiling (SN-ROP) to monitor dynamic changes in redox-related pathways during redox stress. SN-ROP quantifies ROS transporters, enzymes, oxidative stress products and associated signaling pathways to provide information on cellular redox regulation. Applied to diverse cell types and conditions, SN-ROP reveals unique redox patterns and dynamics including coordinated shifts in CD8 T cells upon antigen stimulation as well as variations in CAR-T cell persistence. Furthermore, SN-ROP analysis uncovers environmental factors such as hypoxia and T cell exhaustion for influencing redox balance, and also reveals distinct features in patients on hemodialysis. Our findings thus support the use of SN-ROP to elucidate intricate redox networks and their implications in immune cell function and disease.

Article	Year
Cigarette smoking increases the risk of nasopharyngeal carcinoma through the elevated level of IgA antibody against Epstein-Barr virus capsid antigen: A mediation analysis. Cancer medicine\| PubMed ID: 31925935	2020
Age effect on in vitro fertilization pregnancy mediated by anti-Mullerian hormone (AMH) and modified by follicle stimulating hormone (FSH). BMC pregnancy and childbirth\| PubMed ID: 32272911	2020
Estimating the causal effect of embryo transfer day on clinical in vitro fertilization outcomes using propensity score matching. BMC pregnancy and childbirth\| PubMed ID: 34388966	2021
Effectiveness of entecavir vs tenofovir disoproxil fumarate for functional cure of chronic hepatitis B in an international cohort. Hepatology international\| PubMed ID: 36070123	2022
Mechanism and modeling of human disease-associated near-exon intronic variants that perturb RNA splicing. Nature structural & molecular biology\| PubMed ID: 36303034	2022
Hypertension as a Novel Link for Shared Heritability in Age at Menarche and Cardiometabolic Traits. The Journal of clinical endocrinology and metabolism\| PubMed ID: 36810613	2023
Immunometabolic features of natural killer cells are associated with infection outcomes in critical illness. Frontiers in immunology\| PubMed ID: 38426112	2024
SpliceAPP: an interactive web server to predict splicing errors arising from human mutations. BMC genomics\| PubMed ID: 38877417	2024
Multiplexed assays of human disease-relevant mutations reveal UTR dinucleotide composition as a major determinant of RNA stability. eLife\| PubMed ID: 39964837	2025
Single-cell signaling network profiling during redox stress reveals dynamic redox regulation in immune cells. Nature communications\| PubMed ID: 40593569	2025

Jia-Ying Su

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