Molecular Genetics and Genomics

Chromatin profiling methods, such as ChIP-seq (chromatin immunoprecipitation followed by sequencing), are used to characterize the genomic localization of DNA-associated proteins. While ChIP and ChIP-seq have been used for decades, an orthogonal approach, CUT&Tag (Cleavage Under Targets and Tagmentation), is gaining popularity as an efficient and cost-effective alternative. Although previous comparative studies note discrepancies in signal-to-noise ratios and detection bias at certain genomic regions, many differences between ChIP-seq and CUT&Tag results remain largely unreconciled. Here, we systematically investigate the disagreeing signals captured by these two methods across well annotated genomic regions. We assess multiple histone mark profiles generated by different groups in two cell lines (K562 and MCF-7). Overall, our analysis indicates that compared to ChIP-seq, CUT&Tag may have limited sensitivity in low-GC environments and, as previously observed, increased signal in hyper-accessible chromatin. Notably, within GC-poor regions of active gene bodies and Polycomb-repressed domains, CUT&Tag exhibits a loss of H3K36me3 and H3K27me3 signal, respectively, where occupancy of these histone marks is otherwise expected. Further, active promoters with discrepant H3K4me3 and H3K27ac signal between the two assays differ systematically in GC content and chromatin accessibility. Promoters differentially enriched for CUT&Tag signal relative to ChIP-seq generally show higher, broader GC-content profiles and higher DNase-seq and ATAC-seq signal, while promoters enriched for ChIP-seq signal harbor the opposite characteristics. A local bias for high GC content and/or chromatin accessibility in CUT&Tag may also explain its differing signal patterns at nucleosome-depleted regions (NDRs) compared to ChIP-seq and MNase-seq promoter profiles. Altogether, our results highlight that studies focused on profiling the intensity and structure of histone modification occupancy can be sensitive to potential biases of CUT&Tag to GC content and chromatin accessibility. These characteristics should be accounted for when selecting a chromatin profiling approach, analyzing and interpreting data as well as drawing biological conclusions.

We applied the latest RIC-seq technology to study the RNA-RNA interactions in human liver HepG2 cells. Integrating ChIP-seq data with the RIC-seq data, we clas-sified vari ous transcription factors (TFs) into two distinct clusters based on the in-teraction enrichment scores of the uaRNA (RNA transcribed in promoter regions) and eRNA (RNA tran scribed in enhancer regions) potentially transcribed by the TF binding sites. We went on to compare the genomic features and clinical significance across different clusters, by further integrating Hi-C, ATAC-seq, and clinical data from liver cancer patients. Our results showed that Cluster 1 exhibited low spatial interaction (RIC-seq derived RNA-RNA interactions between regulatory RNA ele-ments) and high tissue specificity (high proportion of binding sites covered by shared peaks between two different cell lines for each histone modification marker), showed higher associations with the B compartments and nucleoli, contained less densely clustered TF binding sites, and is more correlated with clinical outcomes. In contrast, Cluster 2 has higher spatial interaction, is more likely to be found in the A compartments and nuclear speckles, contained more densely clustered TF binding sites, and is more prone to form loops. These findings suggested that these transcrip-tion factors might play critical roles in regulating gene expression and serve as po-tential biomarkers and therapeutic targets with significant implications for cancer prognosis and treatment. In summary, groups of TFs could occur as distinct sub-networks based on spatial proximity of non-coding RNAs transcribed by the TF binding sites. These sub-networks appear to be related to regulatory differences for the TF groups and may partially explain the chromosome compartments/domains.

Objective This study aimed to develop and validate a predictive model for sleep disorders in young and middle-aged adults with anemia, providing evidence for early identification, risk stratification, and individualized sleep management. Methods Data in the NHANES were analyzed and randomly divided into development and validation cohorts. LASSO regression was used to screen optimal predictors in the development cohort, followed by univariate and multivariate logistic regression to identify independent predictors and construct a nomogram. Model performance was evaluated using receiver operating characteristic curves, calibration curves, Brier scores, and decision curve analysis. Results The weighted prevalence of sleep disorders among young and middle-aged adults with anemia was 10.19%. Multivariate logistic regression identified seven independent predictors: sex, monocyte count, asthma, overweight, arthritis, congestive heart failure, and hypertension. The model showed good discrimination, with area under the curve values of 0.817 in the development cohort and 0.791 in the validation cohort. Calibration was satisfactory, with Brier scores of 0.074 and 0.075, respectively. Decision curve analysis indicated favorable clinical net benefit in both cohorts. Conclusion The proposed predictive model demonstrated good discrimination, calibration, and may help healthcare providers identify young and middle-aged adults with anemia who are at high risk for sleep disorders.

A population-specific missense variant, rs373863828 (G > A, p.Arg457Gln), has been identified in Pacific Islanders and is associated with increased body mass index and lower odds of type 2 diabetes. However, the mechanism underlying these associations is unknown. The variant is located in exon 5 of CREBRF, a gene which is rapidly upregulated in response to nutrient deprivation. This study aimed to assess the effect of rs373863828 on gene expression in adipose tissue from Samoan adults from the Soifua Manuia (''Good Health'') study. Needle subcutaneous adipose (upper buttock) biopsies were obtained in Apia, Samoa, from fasting Samoans without diabetes. RNA isolated from adipose tissue (n=91, 65.9% female) was subjected to bulk paired-end mRNA-sequencing (2x150 base pair). Differential gene expression among the three rs373863828 genotypes (GG, AG, AA) was assessed using a likelihood-ratio test implemented in DESeq2, and overrepresentation analysis for significant differentially expressed genes (DEGs) (padj < 0.1) was performed with clusterProfiler and oRG.DB. Bulk expression was deconvoluted to determine the cell type composition of the adipose tissue. Twenty-two DEGs were associated with rs373863828 genotype including CBX7 (padj=0.026 ) and SELENOM (padj= 0.004), both of which have roles in adipogenesis and metabolism. A single expression profile comprising 13 of the 22 DEGs corresponding to CREBRF genotype was identified, and overrepresentation analysis supported this finding, with terms such as ''keratinocyte differentiation'' and ''skin development'' enriched among significant DEGs. However, PERMANOVA of adipose cell type proportions showed no difference (p > 0.05) among the three genotypes. rs373863828 genotype was associated with expression of 22 genes in whole subcutaneous adipose tissue of this sample of fasting Samoan adults without diabetes. Future work should assess cell- and tissue-specific rs373863828 genotype effects using a combination of single-cell approaches and human cell models with perturbations associated with nutritional stress.

Tsetse flies are strictly found is sub-Saharan Africa where they are responsible for the transmission and maintenance of African trypanosomiases in humans (HAT) and animals (AAT). Vector control has been recognized as an essential tool to fight against these diseases. Nevertheless, it requires the best possible knowledge of the biology of the targeted population. Population genetics tools can prove very useful to obtain such information but require the use of polymorphic and reliable genetic markers. In this paper we present the development of microsatellite markers using a new high-throughput sequencing based technology (SSRseq). We applied it on two species of tsetse flies from three HAT foci and obtained more accurate results as compared to microsatellite loci developed with classic methods. We could indeed use 9 to 14 SSRseq loci without the several problems generally met with classic microsatellites, as all were located in autosomes, without short allele dominance or stuttering and very few null alleles. SSRseq loci appeared much more polymorphic in tsetse flies as compared to other species (fungi, trees, bees, or fishes), which suggested much higher effective population sizes, much higher mutation rates of the genome or both, suggesting high capacities for evolutionary adaptation. With the 9-14 loci kept, we confirmed the propensity of these flies to move almost freely in the whole zones investigated, and also highlighted the possible evolutionary response of one of the unkept loci regarding vector control devices used in these HAT foci, which will require further studies. We suggest for further population structure studies to use only loci with less than 1% missing data, which proved being a good and fast selection strategy to get the most reliable results.

Ancestry-related genomic variation may contribute to phenotypic diversity. However, its cellular consequences remain difficult to evaluate. To address this, we developed a human induced pluripotent stem cell (hiPSC)-based framework for investigating such phenotypes. We used the Japanese population as a model of Jomon-related and continental East Asian ancestry admixture. Jomon ancestry, estimated using the Jomon allele score (JAS), was associated with clinical measures, including renal-related traits and serum triglycerides in 34,152 Tohoku Medical Megabank participants. Peripheral blood mononuclear cell transcriptomics of donors from the upper and lower extremes of the JAS distribution (n = 10) suggested group-level differences in immune-related gene expression. To test whether the serum triglyceride association coincided with hepatic cellular phenotypes, we differentiated hiPSCs from these donors into hepatocyte-like cells. Neither intracellular triglycerides nor lipid-related gene expression differed between groups. These findings highlight the value of a genetically characterized hiPSC-based framework for investigating ancestry-associated phenotypes across cellular contexts.

Since the discovery of the genetic code, a frequently discussed question is whether the numbers of synonymous codons for the various amino acids are randomly distributed or were shaped by evolutionary constraints. In this study, we analyze for the standard as well as alternative genetic codes, the correlations between codon degeneracy and amino acid frequencies in proteins (neglecting differences in gene expression). To taking into account the effect of GC content, expected codon frequencies rather than codon multiplicity need to be considered. A strong correlation of these frequencies with amino acid abundance is revealed. Furthermore, we identify consistent patterns of over- and underrepresentation of amino acids across domains of cellular life as well as viruses. For example, the codons for glutamate, aspartate, lysine, and methionine are consistently overrepresented across domains, while cysteine, arginine, histidine, and proline are underrepresented. Subsequently, we discuss the role of biosynthesis costs of amino acids and other factors such as the order of amino acid recruitment in early evolution, exposure to oxidative stress, and sulphur availability. We hypothesize that in a first phase of evolution, the genetic code evolved in a way so as to comply with the different demands for amino acids. In a second phase, after the code was frozen, changes in demand led to deviations from the strong correlation between codon multiplicity and amino acid frequency by natural selection. This comprehensive analysis offers new insights into the interplay between genetic code structure and amino acid usage across all domains of life.

ABSTRACT Objective: Skeletal muscle growth rate differs substantially among diverse pig breeds. In the present study, RNA-seq was performed to identify differentially expressed genes (DEGs) across pig breeds with distinct growth rates. CCND2 showed obvious differential expression among pig breeds. Although CCND2 is essential for cell cycle control, its biological function in skeletal muscle differentiation has rarely been reported. This work aims to identify DEGs in pig breeds with different growth rates and explore CCND2 role in porcine skeletal muscle development. Methods: RNA-seq was performed to identify differentially expressed genes (DEGs) across pig breeds with distinct growth rates. Weighted gene co-expression network analysis (WGCNA) was used to identify gene modules potentially associated with skeletal muscle growth and development. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis was performed to validate the role of CCND2 in skeletal muscle differentiation. Results We identified 5,416, 4,846, and 2,777 DEGs in the Duroc pig (DU) vs. Jiangquan black pig (JQ), DU vs. Laiwu pigs (LW), and JQ vs. LW comparison groups, respectively. WGCNA identified two gene modules (lightgreen and darkmagenta) potentially associated with skeletal muscle growth and development. CCND2 exhibited differential expression in comparisons between DU vs JQ and DU vs LW, with higher expression observed in Duroc pigs. qRT-PCR analysis revealed that overexpression of CCND2 at day 6 (D6) of cell differentiation significantly increased the expression levels of CCND2 gene and markers of myogenic differentiation (p < 0.05). Conversely, knockdown of CCND2 significantly decreased the expression levels of CCND2 gene and myogenic differentiation markers (p < 0.05). Conclusions This study identified breed-specific DEGs and key gene modules in porcine skeletal muscle and verified that CCND2 promotes myogenic differentiation. These findings provide a genetic theoretical basis for further investigations into skeletal muscle growth and development. Keywords pig, skeletal muscle, transcriptome, WGCNA, CCND2

Chromosome was "self-assembled" into a complex three-dimensional topology in the nucleus, which provided the structural basis for important biological processes such as gene expression regulation and cell differentiation. The current understanding of the three-dimensional organization of the genome is based on high-throughput sequencing and imaging technologies to visualize the conformation of chromatin in its native environment. This study proposes a new research perspective and technical means to use fusion genes to determine the interaction information and three-dimensional structure of chromosomes. This three-dimensional information provided by the fusion gene spectrum not only shows the universality of the spatial layout of chromosomes, but also increases the interpretability of the occurrence of fusion genes. The chromatin conformation capture data of multiple tissues and periods is significantly correlated with the strength of fusion genes. For example, the frequency of trans-interaction between chromosomes 11 and 17 was the highest, and the frequency of fusion gene occurrence corresponding to KMT2A on chromosome 11 was the highest. Therefore, we infer that this structural universality is a common feature of the human genome, which provides a theoretical basis and data for predicting chromosome fusion events.

In eukaryotic cells, the positive transcription elongation factor b (P-TEFb) plays a critical role in the transition of RNAPII from the paused state to actively transcribing mode. Although three different cyclin (Cyc) T (CycT1, T2a, and T2b) interact with CDK9 to form functional P-TEFb, a majority of P-TEFb complexes are comprised of the CycT1 and CDK9. To analyze the role of the CycT1 subunit of P-TEFb on cellular transcription, we established HEK293T cells lacking CycT1 proteins (CycT1-KO cells). While no apparent growth defects were observed with CYcT1-KO cells, the level of CDK9 was decreased and no compensatory over-expression of CycT2 was observed, suggesting that the reduced level of functional P-TEFb complexes is sufficient for the normal cell growth. Reporter gene assays indicate that NF-kappaB-dependent transcription induced by PMA was impaired in CycT1-KO cells although the function of NF-kappaB (P65) per se was not affected. On the other hand, AP1-dependent transactivation induced by PMA was unaffected in CycT1-KO cells. Moreover, transcription stimulated by JQ1, a strong P-TEFb inducer, was reduced in CycT1-KO cells. Data of biochemical analysis indicate that P-TEFb with CycT1 (P-TEFb (CDK9:CycT1)) was efficiently released from 7SKsnRNP by JQ1 while P-TEFb (CDK9:CycT2) was not affected by JQ1. Transcriptome analysis indicated that the lack of CycT1 had a minor effect on the steady-state transcription. However, expression of JQ1-dependent genes was severely reduced in CycT1-KO cells. Interestingly, referring to gene expression profiles of 33 different types of cancer revealed that 11 out of 13 top JQ1-dependent genes were also highly upregulated in Pancreatic Adenocarcinoma. From these results, we conclude that although many genes can be regulated by both P-TEFb (CDK9:CycT1) and P-TEFb (CDK9:CycT2), CycT1 plays a critical role in regulating highly inducible genes, which are also aberrantly regulated in a particular type of cancer.

Background Fibromyalgia syndrome (FMS) affects 1 to 2 percent of the general population, with higher prevalence estimates in women. Recent evidence supports peripheral immune involvement in disease pathophysiology. Alternative splicing (AS) regulates immune cell function independently of transcript abundance and carries disease-relevant signal in related autoimmune conditions. No study has performed transcriptome-wide differential AS analysis directly in RNA-sequencing data from individuals with FMS. Methods We performed a secondary analysis of GSE221921, restricted to female participants (n = 91 FMS, n = 41 controls). Peripheral immune cell composition was estimated with ABIS, a deconvolution method specifically trained on peripheral blood mononuclear cell signatures. Differential AS was analysed with LeafCutter incorporating cell composition principal components as covariates. Differential gene expression was performed with DESeq2 under the same adjustment framework. Results FMS samples showed lower ABIS-inferred conventional monocyte estimates (Cliff's {delta} = -0.38, FDR = 0.008) and higher memory B-cell estimates (Cliff's {delta} = +0.32, FDR = 0.030). Composition-adjusted LeafCutter analysis identified 14 significant intron clusters, of which 9 were retained for biological interpretation. GNLY (granulysin) was the primary candidate, with a composition-adjusted 5' junction usage shift illustrated by pooled Sashimi visualisation. Composition adjustment substantially reduced significant differentially expressed genes from 10,803 to 3,186 (baseMean [&ge;] 10). Conclusions This first transcriptome-wide differential AS analysis in FMS identifies GNLY, encoding the cytolytic lymphocyte effector granulysin, as a splicing-specific candidate for functional follow-up. AS captures FMS-associated signal not detectable by gene-level expression analysis, and composition adjustment substantially altered the differential expression landscape.

Single cell differential expression analysis enables biologists to make statistical conclusions about which genes are up or downregulated in a particular cell type, between two conditions, such as those with or without a disease. However, due to biological and technical noise, these differences are hard to detect reliably. Determining if an experiment has sufficient power is often derived from simulations or small pilot samples, if done at all. Here, we use sex-biased differential expression on 1,494 donors in three brain cell types to derive empirically grounded power estimates for a variety of experimental setups. Our work reveals a substantial lack of power for reliably detecting the small effects in the range that many studies report, even in experiments containing 600 donors. When reducing the astrocyte data to the poor sequencing characteristics of microglia, over half the differentially expressed genes (DEGs) detected in the full set were lost, highlighting the damage caused by insufficient sequencing depth and cell counts. Despite standard thresholds of adjusted p-value with multiple testing correction, only the top quartile of significant genes were reproducible. Furthermore, from fitting a predictive model to the empirical outcomes, we find cell count and expression levels of genes to be a strong determinant of power. As such, we advocate for future studies to employ cell type enrichment and deeper sequencing, especially for rarer populations like microglia, emphasise the importance of powering experiments of this type when seeking robust findings, and suggest stricter significance thresholds for future discoveries.

Transposable elements (TEs) are major contributors to genomic and regulatory variation and can influence the expression of nearby genes in response to environmental stress. The invasive fruit fly Drosophila suzukii, whose genome is composed of nearly 50% TE-derived sequences, provides an excellent model to investigate the regulatory impact of TEs on stress responses. Although previous studies found no global association between TEs and oxidative stress transcriptomic response in this species, the effects of individual TE insertions remain largely unexplored. Here, we investigated the role of two polymorphic intronic TE insertions, Mrp4-TE and inaE-TE, located within genes implicated in the oxidative stress response in D. melanogaster. Using wild-type strains of D. suzukii, we assessed the effect of these insertions on gene expression following oxidative stress induced by paraquat exposure. We found that their corresponding nearby genes Mrp4 and inaE were upregulated after paraquat treatment and that this response was significantly stronger in flies carrying the corresponding TE insertions. Allele-specific expression analyses further showed that the alleles containing the TE insertions exhibited a tendency towards higher expression levels than alleles lacking the insertions, both under control and oxidative stress conditions, supporting a cis-regulatory effect. In addition, Mrp4-TE was associated with increased sensitivity to oxidative stress. Together, our results provide the first evidence that TE insertions can modulate oxidative stress-responsive gene expression in D. suzukii. These findings highlight the importance of investigating individual TE insertions to understand the genetic and regulatory mechanism underlying stress responses, adaptation, and invasion success in this species.

Parasitoid wasps develop in or on other arthropods and have evolved a wide range of strategies for exploiting their hosts. Ascogaster reticulata is a solitary egg to larval endoparasitoid wasp belonging to the subfamily Cheloninae of Braconidae. We constructed a genome assembly using long-read sequence data. The assembled genome was 280.5 Mbp, consisting of 209 contigs. The N50 value and GC content ratio were 13.9 Mbp and 30.43%, respectively. The percentage of transposable elements and repetitive regions was 52.43%. The predicted number of genes is 27,261. Merqury revealed that the QV value and completeness of the genome were 61.79 and 99.3917%, respectively. BUSCO analysis detected approximately 99% of the core genes. These results suggest that the genome data were of sufficient quality for use as a reference genome. Therefore, these data can be utilized as a genomic resource for comparative studies within Braconidae and for future investigations into the biology and evolution of Cheloninae parasitoid wasps.

Gene model for the ortholog of Insulin-like peptide 3 (Ilp3) in the D. pseudoobscura Apr. 2013 (BCM-HGSC Dpse_3.0/DpseGB3) Genome Assembly (GenBank Accession: GCA_000001765.2) of Drosophila pseudoobscura. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

Melittin, a potent amphipathic cationic peptide derived from bee venom, exhibits broad-spectrum antineoplastic efficacy, notably against cervical carcinoma. Despite its established therapeutic potential, the global transcriptional reprogramming orchestrating its acute multi-pathway cytotoxicity remains incompletely understood. To bridge this knowledge gap, we generated the first comprehensive, untargeted RNA-seq dataset profiling the acute phase of melittin-induced cell death in murine cervical carcinoma U14 cells (exposed to 4 g/mL melittin for 20 minutes) alongside untreated controls. Utilizing deep sequencing and rigorous bioinformatics workflows, we quantified genome-wide mRNA abundances and mapped a distinct transcriptomic shift, identifying 254 significantly differentially expressed genes, comprising 158 up- and 96 down-regulated transcripts. Validated by stringent quality control metrics, exceptional genomic mapping rates, and comprehensive functional annotations via the GO and KEGG databases, this high-resolution transcriptomic resource provides a systems-level map of early molecular alterations. All raw and processed sequencing data are publicly available. This transcriptomic resource provides a valuable foundation for elucidating the acute regulatory networks underlying melittin-induced anti-cervical cancer effects. DatasetThe dataset can be accessed through the NGDC website by searching with the BioProject accession number PRJCA068439. Reviewers may use this link for anonymous access during the review process. Direct URL to data: Genome Sequence Archive-CNCB-NGDC

Trisomy 21 (T21) is the most common genetic cause of intellectual disability, yet the molecular mechanisms underlying maternal meiosis I errors--responsible for ~70% of free T21 cases--remain poorly understood. In this preliminary study, we used long-read sequencing and genome assembly to investigate the DNA sequence and epigenetic features of chromosome 21 (chr21) centromeres in a family with recurrent free T21 due to maternal meiosis I errors. The mother, who had two affected and three unaffected children, showed no mosaicism or structural rearrangements. One of her two chr21 centromeres lacked a pronounced centromere dip region (CDR), displaying instead a diffuse hypomethylation pattern (dCDR) with much higher methylated CpG levels (55%) compared to its homologue (36%). This dCDR was transmitted to an unaffected child and the affected proband analyzed, suggesting it was present in one of the maternal chr21 since she was at least 32 years of age. Chr21 dCDRs were not observed in seven young mothers with children with T21 or previously described in the literature in 108 population haplotypes. We hypothesize that dCDRs may weaken kinetochore function, increasing nondisjunction risk, and propose two models linking such epigenetic variation to maternal age-related T21 risk. These findings highlight the value of complete centromere characterization in families with children with T21 and suggest centromere methylation status of chr21 as a potential T21 risk factor for future investigation.

Gene model for the ortholog of Density regulated protein (DENR) in the Apr. 2013 (BCM-HGSC Dpse_3.0/DpseGB3) Genome Assembly (GenBank Accession: GCA_000001765.2) of Drosophila pseudoobscura. This ortholog was characterized as part of a developing dataset to study the evolution of the Insulin/insulin-like growth factor signaling pathway (IIS) across the genus Drosophila using the Genomics Education Partnership gene annotation protocol for Course-based Undergraduate Research Experiences.

This study aims to quantify the genetic similarity of different species (from fish to humans) to the human reference genome (pp6, Homo sapiens.GRCh38) based on the allele presence/absence patterns of 33 language/cognition related gene SNV loci, identify key breakpoints during evolution, and evaluate the enrichment of language and cognition genes at these breakpoints. We designed a similarity calculation method relying on binary features (four columns for A/T/C/G), adopted five difference/distance measures (Sorensen, Rogers, Nei, Reynolds, and Hellinger), and converted them into similarity values (1/(1+distance)). For each method, samples were independently ranked, the first derivative of similarity was computed, and the top 12 peaks were selected as candidate breakpoints. Results show that the similarity curves from the five methods are highly consistent (correlation coefficients >0.9), with major peaks concentrated at positions 355, 363, 381, 382, 390, 400, etc., where the corresponding samples are predominantly ancient hominins and primates. Furthermore, we defined 13 peak groups (starting positions 355-401). For each peak within a group, pairwise SNV differences between the peak apex sample and its immediate left neighbor were compared, and the intersection F_INTERSECTION (shared differential loci) was obtained. For each F_INTERSECTION, we calculated the proportions of language genes and cognition genes. In addition, we computed the differential sets between adjacent groups' F_INTERSECTION to trace the gradual emergence of new loci. In F_INTERSECTION, language genes accounted for an average of 59.5%, and cognition genes for an average of 62.9%. The proportion of language genes reached a peak at position 383 (61.2%), while cognition genes peaked at position 386 (64.9%). High frequency peak samples include c25, c27, and ja2, suggesting that language cognition genes may have undergone independent intensification during Eurasian evolution. Differential analysis between adjacent F_INTERSECTION revealed a stepwise acquisition of new loci from position 355 to 401, with three bursts of newly added loci along the entire evolutionary axis. This study provides a quantitative framework based on similarity curves, offers a novel molecular perspective for understanding the evolution of language and cognitive abilities, and highlights the potential importance of East Asian archaic hominins in the evolution of language cognition genes.

Preeclampsia (PE) is a hypertensive pregnancy-specific disorder and a leading cause of maternal and fetal mortality. A common feature of PE placentas and maternal plasma is dyslipidemia, or abnormal lipid levels, which can increase oxidative stress and endothelial dysfunction. However, the precise transcriptional, post-transcriptional, and epigenetic mechanisms underlying these abnormalities remain poorly characterized. Identifying such changes may clarify disease mechanisms and identify lipid-related PE biomarkers. We conducted a large-scale meta-analysis integrating public placental datasets from NCBI GEO, comprising four DNA methylation (DNAm) datasets (n = 172), three RNA-sequencing datasets (n = 92), and an independent RNA microarray validation cohort (n =146). We evaluated differential DNAm (limma), gene expression (DESeq2), transcript-level shifts (Swish), and alternative splicing (rMATS) in PE versus control placentas, with all analyses stratified by fetal sex via an interaction term model. We also performed placental cell-type deconvolution to quantify PE-associated cell-type proportion changes. Our results demonstrated that lipid-related regulation changes in PE placentas occur primarily at the gene and transcript level, with DNAm showing no changes. We also identified significant isoform switching in PE that were undetected by differential gene expression analysis, and primarily driven by alternative transcription initiation and termination sites rather than alternative splicing. A subset of these isoform switches mapped to pathways dysregulated in PE and were predicted to cause functional protein changes. An interaction term model identified several sex-specific differentially expressed genes (DEGs) in PE, including a subset of male-specific downregulated genes involved in oxidative metabolism. However, many of the remaining sex-specific DEGs across both sexes were previously uncharacterized in the literature. These findings suggest that transcriptional and isoform-level regulation play a role in PE-associated dyslipidemia, with certain regulatory pathways displaying fetal sex-specific patterns. Highlights- Preeclampsia-associated dyslipidemia manifests at the gene and transcript level - Reciprocal isoform switches were missed by standard gene-level analyses - Alternative transcript initiation and termination drove isoform switching - Sex-interaction modeling identified sex-specific transcriptional shifts in PE