Biology subjects

Lin, J.-H.

Publications and source records attributed to Lin, J.-H..

10 recordsLinked to original sources

bioRxiv2025

When splicing is not all or none: Implications for variant classification

Variants with intermediate functional effects--those that neither eliminate nor fully maintain normal gene function--represent an underrecognized source of genetic complexity and constitute a grey zone that challenges variant classification. Here, we address this issue using GT>GC (+2T>C) 5' splice-site variants as a tractable model, as approximately 15% of such substitutions retain variable amounts of wild-type (WT) transcript. Using residual WT transcript as a quantitative readout, we first show that disease-associated GT>GC variants known to generate substantial WT transcript (for example, SPINK1 c.194+2T>C, HBB c.315+2T>C, and BRCA2 c.8331+2T>C) consistently complicate classification efforts. We then carried out a locus-wide assessment of all 26 GT>GC substitutions in CFTR, applying SpliceAI delta donor-loss scores as an initial screen and cross-checking classifications in expert-curated database. This evaluation indicated that three variants with low or intermediate SpliceAI scores are likely to retain WT transcript. Minigene analyses of four selected CFTR variants, together with additional examination of a BAP1 GT>GC variant with conflicting clinical interpretations, illustrate the methodological challenges in predicting and experimentally quantifying residual WT transcript. These results emphasize that (i) in silico predictors cannot reliably discriminate between complete and partial splice disruption and (ii) experimental systems differ in their ability to detect subtle splicing outcomes. Overall, our integrative analysis shows that GT>GC variants generating appreciable WT transcript exemplify a broader class of intermediate-effect alleles that complicate both computational prediction and experimental assessment, underscoring the need for classification frameworks that incorporate quantitative functional data and better capture the continuum of variant effects.

genetics↗

bioRxiv2024

Antibodies against the capsid induced after intracranial AAV administration limits second administration in a dose dependent manner

Recombinant adeno-associated virus (rAAV) is a widely used viral vector for gene therapy. However, a limitation of AAV-mediated gene therapy is that patients are typically dosed only once. In this study, we investigated the possiblility to deliver multiple rounds of AAV through intracerebral injections in the mouse brain. We discovered a dose-dependent modulation of the second round AAV infection by the first round AAV injection in the brain-wide scales besides the injection region. High-dose AAV infection increases chemokines CXCL9 and CXCL10 to recruit the parenchymal infiltration of lymphocytes. Surprisingly, the blood-brain-barrier was relatively intact. Brain-wide dissection discovered the likely rountes of the infiltrated lymphocytes through perivascular space and ventricles. Further analysis using B-cell depleted mice revealed that B lymphocytes, but not T lymphocytes, played a critical role in inhibiting the second round AAV infection. Strategies against neutralizing antibodies had limited effects, while reducing the dosage for the first injection or switching the second AAV to a different serotype appeared to be more effective in antagonizing the first round AAV inhibition. Together, these results suggest that mammalian brains are not immunoprivileged for AAV infection, but multiple rounds of AAV gene therapy are still possible if designed carefully with proper doses and serotypes.

immunology↗

bioRxiv2022

Efficient Dlx2-mediated astrocyte-to-neuron conversion and inhibition of neuroinflammation by NeuroD1

In vivo astrocyte-to-neuron (AtN) conversion induced by overexpression of neural transcriptional factors has great potential for neural regeneration and repair. Here, we demonstrate that a single neural transcriptional factor Dlx2 converts mouse striatal astrocytes into neurons in a dose-dependent manner. Lineage-tracing studies in Aldh1l1-CreERT2 mice confirm that Dlx2 can convert striatal astrocytes into DARPP32+ and Ctip2+ medium spiny neurons (MSNs). Time-course studies reveal a gradual conversion from astrocytes to neurons in 1 month, with a distinct intermediate state in-between astrocytes and neurons. Interestingly, when Dlx2-infected astrocytes start to lose astrocytic markers, the other local astrocytes proliferate to maintain astrocytic level in the converted areas. Unexpectedly, while Dlx2 efficiently reprograms astrocytes into neurons in the grey matter striatum, it also induces partial reprogramming of astrocytes in the white matter corpus callosum. Such partial reprogramming of white matter astrocytes is associated with neuroinflammation, which can be essentially suppressed by the addition of NeuroD1. Our results highlight the importance of investigating AtN conversion both in the grey matter and white matter in order to thoroughly evaluate therapeutic potentials. This study also unveils a critical role of anti-inflammation by NeuroD1 during AtN conversion.

neuroscience↗

bioRxiv2022

The 24-chain core-shell nanostructure of wood cellulose microfibrils in seed plants

Wood cellulose microfibrils (CMFs) are the most abundant organic substance on earth, but their nanostructures are poorly understood. There are controversies regarding the glucan chain number (N) of CMFs during initial synthesis and whether they become fused afterwards. Here, we combined small-angle X-ray scattering (SAXS), solid-state nuclear magnetic resonance (ssNMR) and X-ray diffraction (XRD) analyses to resolve these controversies. We successfully developed SAXS measurement methods for the cross-section aspect ratio and area of the crystalline-ordered CMF core, which showed higher density than the semi-disordered shell. The 1:1 aspect ratio suggested that CMFs remain mostly segregated, not fused. The area measurement revealed the chain number in the core zone (Ncore). The ratio of ordered cellulose over total cellulose, termed Roc, was determined by ssNMR. Using the formula N = Ncore / Roc, we found that the majority of wood CMFs contain 24 chains, conserved between gymnosperm and angiosperm trees. The average wood CMF has a crystalline-ordered core of [~]2.2 nm diameter and a semi-disordered shell of [~]0.5 nm thickness. In naturally and artificially aged wood, we only observed CMF aggregation (contact without crystalline continuity) but not fusion (forming conjoined crystalline unit). This further argued against the existence of partially fused CMFs in new wood, overturning the recently proposed 18-chain fusion hypothesis. Our findings are important for advancing wood structural knowledge and more efficient utilization of wood resources in sustainable bio-economies.

plant biology↗

bioRxiv2021

LongPhase: an ultra-fast chromosome-scale phasing algorithm for small and large variants

Long-read phasing has been used for reconstructing diploid genomes, improving variant calling, and resolving microbial strains in metagenomics. However, the phasing blocks of existing methods are broken by large Structural Variations (SVs), and the efficiency is unsatisfactory for population-scale phasing. This paper presents an ultra-fast algorithm, LongPhase, which can simultaneously phase single nucleotide polymorphisms (SNPs) and SVs of a human genome in [~]10-20 minutes, 10x faster than the state-of-the-art WhatsHap and Margin. In particular, LongPhase produces much larger phased blocks at almost chromosome level with only long reads (N50=26Mbp). We demonstrate that LongPhase combined with Nanopore is a cost-effective approach for providing chromosome-scale phasing without the need for additional trios, chromosome-conformation, and single-cell strand-seq data.

bioinformatics↗

bioRxiv2020

Identification of 38 novel loci for systemic lupus erythematosus and genetic heterogeneity that may underly population disparities in this disease

Systemic lupus erythematosus (SLE), a worldwide autoimmune disease with high heritability, shows differences in prevalence, severity and age of onset among different ancestral groups. Previous genetic studies have focused more on European populations, which appear to be the least affected. Consequently, the genetic variations that underly the commonalities, differences and treatment options in SLE among ancestral groups have not been well elucidated. To address this, we undertook a genome-wide association study, increasing the sample size of Chinese populations to the level of existing European studies. Thirty-eight novel SLE-associated loci and incomplete sharing of genetic architecture were identified. Nine disease loci showed clear ancestral group heterogeneity and implicated antibody production as a potential mechanism for differences in disease manifestation. Polygenic risk scores performed significantly better when trained on matched ancestral data sets. These analyses help to reveal the genetic bases for disparities in SLE among ancestral groups.

genetics↗

bioRxiv2019

The experimentally obtained functional impact assessments of GT>GC 5' splice site variants differ markedly from those predicted

GT>GC 5 splice site (or +2T>C) variants have been frequently reported to cause human genetic disease. However, although we have demonstrated that GT>GC variants in human disease genes may not invariably be pathogenic, none of the currently available splicing prediction tools appear to be capable of reliably distinguishing those GT>GC variants that generate wild-type transcripts from those that do not. Recently, SpliceAI, a novel deep residual neural network tool, has been developed for splicing prediction. Methodologically distinct from previous approaches that either rely on human-engineered features and/or which focus on short nucleotide windows adjoining exon-intron boundaries, SpliceAI assesses splicing determinants by evaluating 10,000 nucleotides of flanking contextual sequence to predict the functional role in splicing of each position in the pre-mRNA transcript. Herein, we evaluated the performance of SpliceAI in the context of three datasets of GT>GC variants, all of which had been characterized functionally in terms of their impact on mRNA splicing. The first two datasets refer to our recently described "in vivo" dataset of 45 disease-causing GT>GC variants and the "in vitro" dataset of 103 GT>GC substitutions. The third dataset comprised 12 BRCA1 GT>GC variants that were recently analyzed by saturation genome editing. We processed all GT>GC variants using the default settings of SpliceAI. Comparison of the SpliceAI-predicted and experimentally obtained functional impact assessments of the analyzed GT>GC variants revealed that although SpliceAI performed rather better than other prediction tools, it was still far from perfect. A key issue is that the impact of GT>GC (as well as GT>GA or +2T>A) variants that generated wild-type transcripts represents a quantitative change that can vary from barely detectable to almost full expression of wild-type transcripts, with wild-type transcripts often co-existing with aberrantly spliced transcripts. Our findings highlight the challenges that we still face in attempting to accurately identify splice-altering variants.

genetics↗

bioRxiv2019

5’ splice site GC>GT variants differ from GT>GC variants in terms of their functionality and pathogenicity

In the human genome, most 5 splice sites (~99%) employ the canonical GT dinucleotide whereas a small minority (~1%) use the non-canonical GC dinucleotide. The functionality and pathogenicity of 5 splice site GT>GC (i.e., +2T>C) variants have been extensively studied but we still know very little about 5 splice site GC>GT (+2C>T) variants. Herein, we sought to address this deficiency by performing a meta-analysis of identified +2C>T pathogenic variants together with a functional analysis of +2C>T substitutions using a cell culture-based full-length gene splicing assay. Our results establish a proof of concept that +2C>T variants are qualitatively different from +2T>C variants in terms of their functionality and pathogenicity and suggest that, in sharp contrast with +2T>C variants, most if not all +2C>T variants have no pathological relevance. Our findings have important implications for interpreting the clinical relevance of +2C>T variants but might also improve our understanding of the evolutionary basis of switching between GT and GC 5 splice sites in mammalian genomes.

genetics↗

bioRxiv2019

ARABIDOPSIS NITRATE REGULATED 1 acts as a negative modulator of seed germination by activating ABI3 expression

Seed germination is a crucial transition point in plant life and is tightly regulated by environmental conditions through the coordination of two phytohormones, gibberellin and abscisic acid (ABA). To avoid unfavorable conditions, plants have evolved safeguard mechanisms for seed germination. Here, we report a novel function of the Arabidopsis MADS-box transcription factor ARABIDOPSIS NITRATE REGULATED 1 (ANR1) in seed germination. ANR1 knockout mutant is insensitive to ABA, salt, and osmotic stress during the seed germination and early seedling development stages, whereas ANR1-overexpressing lines are hypersensitive. ANR1 is responsive to ABA and abiotic stresses and upregulates the expression of ABI3 to suppress seed germination. ANR1 and ABI3 have similar expression pattern during seed germination. Genetically, ABI3 acts downstream of ANR1. Chromatin immunoprecipitation and yeast-one-hybrid assays showed that ANR1 could bind to the ABI3 promoter to regulate its expression. In addition, ANR1 acts synergistically with AGL21 to suppress seed germination in response to ABA as evidenced by anr1 agl21 double mutant. Taken together, our results demonstrate that the ANR1 plays an important role in regulating seed germination and early post-germination growth. ANR1 and AGL21 together constitutes a safeguard mechanism for seed germination to avoid unfavorable conditions.

plant biology↗

bioRxiv2018

First estimation of the scale of canonical 5’ splice site GT>GC mutations generating wild-type transcripts and their medical genetic implications

It has long been known that canonical 5 splice site (5SS) GT>GC mutations may be compatible with normal splicing. However, to date, the true scale of canonical 5SS GT>GC mutations generating wild-type transcripts, both in the context of the frequency of such mutations and the level of wild-type transcripts generated from the mutation alleles, remain unknown. Herein, combining data derived from a meta-analysis of 45 informative disease-causing 5SS GT>GC mutations (from 42 genes) and a cell culture-based full-length gene splicing assay of 103 5SS GT>GC mutations (from 30 genes), we estimate that [~]15-18% of the canonical GT 5SSs are capable of generating between 1 and 84% normal transcripts as a consequence of the substitution of GT by GC. We further demonstrate that the canonical 5SSs whose substitutions of GT by GC generated normal transcripts show stronger complementarity to the 5 end of U1 snRNA than those sites whose substitutions of GT by GC did not lead to the generation of normal transcripts. We also observed a correlation between the generation of wild-type transcripts and a milder than expected clinical phenotype but found that none of the available splicing prediction tools were able to accurately predict the functional impact of 5SS GT>GC mutations. Our findings imply that 5SS GT>GC mutations may not invariably cause human disease but should also help to improve our understanding of the evolutionary processes that accompanied GT>GC subtype switching of U2-type introns in mammals.

genetics↗