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Chen, J.-M.

Publications and source records attributed to Chen, J.-M..

16 recordsLinked to original sources

Context-dependent variant interpretation from Mendelian disease to genetic predisposition: a proof-of-concept using LPL

As genomic sequencing evolves beyond rare disease diagnostics toward population screening and precision medicine, clinical variant interpretation is increasingly challenged by variants whose clinical consequences depend on biological context. Current frameworks, including the ACMG/AMP guidelines, generally assign a single classification to each variant regardless of inheritance state or genetic context, potentially failing to communicate context-dependent clinical consequences. Here, we address this issue using loss-of-function variants in LPL as a uniquely informative model system in which residual physiological LPL activity can be directly quantified in vivo. By systematically integrating published biallelic LPL genotypes, physiological measurements, functional studies, and clinical phenotypes, we identified a biologically meaningful transition at approximately 10% residual physiological LPL activity. Activity below this level was predominantly associated with classical childhood-onset familial chylomicronemia syndrome (FCS), whereas higher activity was associated with phenotypic attenuation and modifier-dependent clinical expression. Furthermore, heterozygous loss-of-function variants exhibited an estimated penetrance of 5-7% for severe hypertriglyceridemia. We therefore propose a context-dependent framework in which biallelic complete- or near-complete loss-of-function genotypes are interpreted as causative for FCS, whereas heterozygous variants are interpreted as predisposing to severe hypertriglyceridemia while retaining recognition of FCS carrier status. Together, our findings demonstrate that clinical variant interpretation should integrate available biological context--including, where relevant, allelic configuration, residual biological function, and penetrance--rather than rely on the intrinsic molecular consequence of the variant alone. More broadly, this framework provides a conceptual model for interpreting variants across the continuum from Mendelian disease to genetic predisposition in the era of precision medicine.

genetics↗

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↗

Natural selection and neutral mutations through the lens of viruses

The Neutral Theory and the Modern Synthesis, a modified version of Darwins theory, have been arguing for decades about the influence of natural selection on molecular evolution1-10. Here we elucidate through the lens of viruses that a frequently used method11-17 employing the ratio of nonsynonymous versus synonymous substitution rates has dramatically underestimated the influence of natural selection on molecular evolution. We also find novel evidence from viral sequences to support the co-existence of the crucial role of natural selection in molecular evolution and the ubiquity of neutral mutations. The co-existence has perplexed biologists for decades2,5,7. We then elucidate for the first time the causality between natural selection and the ubiquity of neutral mutations with a novel interpretation of natural selection. This novel interpretation incorporates biochemistry, genetics, epigenetics, physiology, and dynamics. It holds that natural selection acts directly on the overall phenotypic performance of organisms and indirectly on each genomic site or phenotypic trait. It highlights not only restrictions and competitions but also freedom and diversity, besides the overall harmonious development of organisms and human societies. Therefore, this novel interpretation could have far-reaching implications in the natural and social sciences.

evolutionary biology↗

Origins of monkeypox viruses and their strains circulating in 2022

Monkeypox has spread unprecedentedly to nearly 100 countries and infected more than 51,000 people since 1 May 2022. This large-scale outbreak constituted a public health emergency of international concern (PHEIC), as declared by the World Health Organization. To better recognize and control this outbreak, we explore here through phylogenetic analysis the origins of MPXVs and their strains circulating in 2022, which remain unclear so far. Our results suggest that MPXVs possibly originated from some cowpox viruses and three lineages of MPXVs within Clade IIb circulated in 2022. Our results also suggest that two MPXVs respectively similar to the two MPXVs exported from Nigeria to the USA in 2021, ON676708/USA/2021 and ON676707/USA/2021, evolved into two lineages and sparked the large-scale outbreak in 2022, after their unknown evolutionary and epidemiological journeys possibly in Nigeria, the USA, or other countries before May 2022. This view does not stigmatize any country, because monkeypox is an endemic zoonosis in west Africa with wildlife reservoirs, and its large-scale outbreak in 2022 also resulted from the global decline of population immunity against smallpox due to the cease of vaccination against smallpox decades ago.

microbiology↗

Frequencies and Features of Frameshift Mutations in Microevolution and Macroevolution of Viruses

Some nucleotide insertions or deletions (indels) in open reading frames of virus genes lead to frameshift mutations (FSMs), which can drastically change amino acid sequences. Most FSMs are deleterious and inhibited by natural selection, but some FSMs could aid viruses in adapting to new hosts, escaping immunity, or changing viral pathogenicity or transmissibility. Surprisingly, various fundamental aspects of FSMs in virus evolution remain unknown. In this report, we identified 679 FSMs in the genomes of viruses from 13 randomly selected animal virus families using randomly selected sequences. The 679 FSMs were formed with 1-5 indels, and most (89.4%) FSMs were fixed through the compensatory mechanism with two indels. Each FSM changed 3-209 (mean=15.4, median=13) amino acid residues. FSM frequencies were usually higher in viral sequences with lower sequence identities and steeply increased when sequence identities declined to 60.0%-69.9% or 70.0%-79.9%. This suggests that FSMs are more important to the macroevolution (i.e., inter-species evolution, including speciation) than to the microevolution (i.e., intra-species evolution) of viruses. This study provided novel evidence for the hopeful monster hypothesis in evolutionary biology. Furthermore, we found that FSMs occurred at different frequencies among genes in the same virus genomes and among virus families. Collectively, this study revealed multiple fundamental features of FSMs in virus evolution for the first time and provided novel insights into the mechanisms of macroevolution and speciation.

evolutionary biology↗

Secondary reversion to sexual monomorphism associated with tissue-specific loss of doublesex expression

Animal evolution is characterized by frequent turnover of sexually dimorphic traits - new sex- specific characters are gained, and some ancestral sex-specific characters are lost, in many lineages. In insects, sexual differentiation is predominantly cell-autonomous and depends on the expression of the doublesex (dsx) transcription factor. In most cases, cells that transcribe dsx have the potential to undergo sex-specific differentiation, while those that lack dsx expression do not. Consistent with this mode of development, comparative research has shown that the origin of new sex-specific traits can be associated with the origin of new spatial domains of dsx expression. In this report, we examine the opposite situation - a secondary loss of the sex comb, a male-specific grasping structure that develops on the front legs of some drosophilid species. We show that, while the origin of the sex comb is linked to an evolutionary gain of dsx expression in the leg, sex comb loss in a newly identified species of Lordiphosa (Drosophilidae) is associated with a secondary loss of dsx expression. We discuss how the developmental control of sexual dimorphism affects the mechanisms by which sex-specific traits can evolve.

evolutionary biology↗

Integrating transcriptomes to investigate genes associated with adaptation to aquatic environments, and assess phylogenetic conflict and whole-genome duplications in Alismatales

Land plants first evolved from freshwater algae, and flowering plants returned to water as early as the Cretaceous and multiple times subsequently. Alismatales is the largest clade of aquatic angiosperms including all marine angiosperms, as well as terrestrial plants. We used Alismatales to explore plant adaptation to aquatic environments by analyzing a data set that included 95 samples (89 Alismatales species) covering four genomes and 91 transcriptomes (59 generated in this study). To provide a basis for investigating adaptation, we assessed phylogenetic conflict and whole-genome duplication (WGD) events in Alismatales. We recovered a relationship for the three main clades in Alismatales as (Tofieldiaceae, Araceae) + core Alismatids. We also found phylogenetic conflict among the backbone of the three main clades that could be explained by incomplete lineage sorting and introgression. Overall, we identified 18 putative WGD events across Alismatales. One of them occurred at the most recent common ancestor of core Alismatids, and three occurred at seagrass lineages. We also found that lineage and life-form were both important for different evolutionary patterns for the genes related to freshwater and marine adaptation. For example, several light or ethylene-related genes were lost in the seagrass Zosteraceae, but are present in other seagrasses and freshwater species. Stomata-related genes were lost in both submersed freshwater species and seagrasses. Nicotianamine synthase genes, which are important in iron intake, expanded in both submersed freshwater species and seagrasses. Our results advance the understanding of the adaptation to aquatic environments and whole-genome duplications using phylogenomics.

evolutionary biology↗

Revision of the genus Dichaetophora Duda (Diptera: Drosophilidae), part I: DNA bar-coding and molecular phylogenetic reconstruction

The genus Dichaetophora is currently comprised of 67 formally described species assigned into five species groups, i.e., agbo, tenuicauda, acutissima, sinensis and trilobita. In the present study, we challenged to delimit species from a huge amount of samples of Dichaetophora and allied taxa (the genus Mulgravea and the subgenus Dudaica of Drosophila) collected from a wide range of the Oriental and east Palearctic regions. We first sorted all specimens into morpho-species, which were tentatively classified into the species groups of Dichaetophora, Mulgravea and Dudaica based on the morphological diagnoses for these supraspecific taxa. Then, we selected representative specimen(s) for each morpho-species and subjected them to barcoding of COI (the cytochrome c oxidase subunit I gene) sequences. The applied ABGD (automatic barcode gap discovery) algorithm estimated a total of 222 MOTUs (molecular operational taxonomic units). Although most of the morpho-species were recognized as individual MOTUs, some others were divided into multiple MOTUs or combined into a single MOTU, suggesting the existence of cryptic sibling species, subspecies or intraspecific genetic varieties. Out of the 222 MOTUs, 88 representing the supraspecific taxa of Dichaetophora, Mulgravea and Dudaica were selected, along with 33 species from major genera and subgenera of Drosophila in the tribe Drosophilini, as in-group (four species from the tribe Colocasiomyini as out-group) for phylogenetic reconstruction. We analyzed a dataset of concatenated nucleotide sequences of 12 nuclear gene markers by the maximum likelihood and Bayesian inference methods. As a result, the three focal taxa (i.e., Dichaetophora, Mulgravea and Dudaica) formed a clade, which we called the "pan-Dichaetophora". Within this large clade, the agbo, tenuicauda, sinensis and trilobita groups of Dichaetophora, Mulgravea and Dudaica were recovered as monophyletic groups, but Dichaetophora and its acutissima group were regarded as paraphyletic. In addition, two clusters were recognized among ungrouped MOTUs of Dichaetophora. Thus, the present study has uncovered some problems in the taxonomy of the pan-Dichaetophora. Solving such problems will be challenged in Part II of this serial work.

zoology↗

Optogenetic Amplification Circuits for Light-Induced Metabolic Control

Dynamic control of microbial metabolism is an effective strategy to improve chemical production in fermentations. While dynamic control is most often implemented using chemical inducers, optogenetics offers an attractive alternative due to the high tunability and reversibility afforded by light. However, a major concern of applying optogenetics in metabolic engineering is the risk of insufficient light penetration at high cell densities, especially in large bioreactors. Here, we present a new series of optogenetic circuits we call OptoAMP, which amplify the transcriptional response to blue light by as much as 21.8-fold compared to the basal circuit (OptoEXP). These circuits show as much as a 41-fold induction between dark and light conditions, efficient activation at light doses as low as ~1%, and strong homogeneous light-induction in bioreactors of at least 5L, with limited illumination at cell densities above 40 OD600. We demonstrate the ability of OptoAMP circuits to control engineered metabolic pathways in novel three-phase fermentations using different light schedules to control enzyme expression and improve production of lactic acid, isobutanol, and naringenin. These circuits expand the applicability of optogenetics to metabolic engineering.

synthetic biology↗

RNA secondary structure mediated by Alu insertion as a novel disease-causing mechanism

We previously identified a homozygous Alu insertion variant (Alu_Ins) in the 3-UTR of the SPINK1 gene as the cause of a novel pediatric disease. Although we established that Alu_Ins leads to the complete loss of SPINK1 mRNA expression, the precise mechanisms remained elusive. Here we aimed to elucidate these mechanisms through a hypothesis-driven approach. Initially, we speculated that Alu_Ins could independently disrupt mRNA 3 end formation and/or affect other post-transcriptional processes such as nuclear export and translation, due to its particular location. However, the presence of Alu_Ins in the 3-UTR resulted in only an ~50% reduction in luciferase reporter activity compared to the wild-type, suggesting the involvement of additional mechanisms. Using RepeatMasker, we identified two Alu elements within SPINK1s third intron, both of which resided in an orientation opposite to that of Alu_Ins. Through RNAfold predictions and full-length gene expression assays designed to examine orientation-dependent interactions between Alu repeats, we present evidence linking the detrimental effect of Alu_Ins to extensive double-stranded RNA structures formed between Alu_Ins and pre-existing intronic Alu sequences. Our results reveal a novel pathogenetic mechanism involving an Alu insertion, highlighting the importance of considering interactions between new and pre-existing Alu elements in inverted orientations within disease-associated genes.

genetics↗

p.E152K-STIM1 mutation deregulates Ca2+ signaling contributing to chronic pancreatitis.

Since deregulation of intracellular Ca2+ can lead to intracellular trypsin activation and STIM1 (stromal interaction molecule-1) protein is the main regulator of Ca2+ homeostasis in pancreatic acinar cells, we explored the Ca2+ signaling in 37 STIM1 variants found in three pancreatitis patient cohorts. Extensive functional analysis of one particular variant, p.E152K, identified in three patients, provided a plausible link between dysregulated Ca2+ signaling within pancreatic acinar cells and chronic pancreatitis susceptibility. Specifically, p.E152K, located within the STIM1 EF-hand and sterile -motif domain, increased the release of Ca2+ from the endoplasmic reticulum in patient-derived fibroblasts and transfected HEK293T cells. This event was mediated by altered STIM1-sarco/endoplasmic reticulum calcium transport ATPase (SERCA) interactions and enhanced SERCA pump activity leading to increased Store Operated Calcium Entry (SOCE). In the pancreatic AR42J cells expressing the p.E152K variant, Ca2+-signaling perturbations correlated with defects in trypsin activation and secretion, and increased cytotoxicity after cholecystokinin stimulation. Summary statementp.E152K-STIM1 variant found in pancreatitis patients leads to intracellular changes in calcium homeostasis through SERCA interaction, enabling intracellular trypsin activation and pancreatic acinar cell death.

cell biology↗

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↗

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↗

Genetic analysis of the STIM1 gene in chronic pancreatitis

Chronic pancreatitis is a complex disease that involves many factors, both genetic and environmental. Over the past two decades, molecular genetic analysis of five genes that are highly expressed in human pancreatic acinar cells, namely PRSS1, PRSS2, SPINK1, CTRC and CTRB1/CTRB2, has established that a trypsin-dependent pathway plays a key role in the etiology of chronic pancreatitis. Since Ca2+ deregulation can lead to intracellular trypsin activation in experimental acute pancreatitis, we analyzed STIM1 (encoding stromal interaction molecule-1, the main regulator of Ca2+ homeostasis in pancreatic acinar cells) as a candidate modifier gene in French, German and Chinese patients with chronic pancreatitis. The French and German subjects were analyzed by Sanger sequencing whereas the Chinese subjects were analyzed by targeted next-generation sequencing confirmed by Sanger sequencing. A total of 37 rare coding variants (35 missense and 2 nonsense) were identified, which were enriched in patients as compared with controls [2.28% (47/2,057) vs. 0.99% (33/3,322); odds ratio = 2.33, P = 0.0001]. This is the first large case-control study to demonstrate a putative association of rare STIM1 coding variants with chronic pancreatitis. Functional analysis will be required to clarify whether or not the rare STIM1 variants detected predispose to pancreatitis.

genetics↗

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↗