Genes

20 pages, 4341 KB

Open AccessArticle

Evaluation of Long-Read RNA Sequencing Procedures for Novel Isoform Identification and Quantification in Human Whole Blood

by Hikari Okada, Alessandro Nasti, Yoshio Sakai, Yumie Takeshita, Sadahiro Iwabuchi, Ho Yagi, Tomomi Hashiba, Noboru Takata, Taka-Aki Sato, Takeshi Urabe, Seiji Nakamura, Toshinari Takamura, Taro Yamashita, Takuro Tamura, Kenichi Matsubara and Shuichi Kaneko

Genes 2025, 16(9), 1075; https://doi.org/10.3390/genes16091075 - 12 Sep 2025

Abstract

Background/Objectives: Blood flows through the body and reaches all tissues, contributing to homeostasis and physiological functions. Providing information and understanding on how the transcriptome of whole blood behaves in response to physiological or pathological stimuli is critical. Methods: We collected blood from four [...] Read more.

Background/Objectives: Blood flows through the body and reaches all tissues, contributing to homeostasis and physiological functions. Providing information and understanding on how the transcriptome of whole blood behaves in response to physiological or pathological stimuli is critical. Methods: We collected blood from four healthy individuals and performed long-read RNA sequencing (lrRNA-seq) for the precise identification and expression quantification of RNA variants. Moreover, we compared two genome references: the Genome Reference Consortium Human Build 38 (GRCh38) and the Telomere-to-Telomere (T2T) assembly of the CHM13 cell line (T2T-CHM13). Results: With GRCh38, we could identify an average of about 46,000 genes, 1.3-fold more genes than T2T-CHM13. Similarly, we identified about 185,000 isoforms with GRCh38 and 140,000 with T2T-CHM13, finding similar differences for full splice match (FSM) and incomplete splice match (ISM) transcript isoforms. There were about 90,000 novel isoforms for GRCh38 and 70,000 for T2T-CHM13, 47% and 50% of the total number of identified isoforms, respectively. Differences in isoform numbers between GRCh38 and T2T-CHM13 were identified for the subcategories “Genic Genomic”, “Intergenic”, and “Genic Intron”. Using GRCh38, we generally identified a higher number of non-coding isoforms, as well as a higher number of isoforms aligning within intron and intergenic regions. Nonetheless, GRCh38 might incur false positive results, and T2T-CHM13 is likely more accurate for genome sequences in the repetitive regions. Conclusions: LrRNA-seq is a valid method for the identification of novel isoforms in blood, and this study is a first step toward the creation of a comprehensive database of the structure and expression of transcript isoforms for optimized predictive medicine. Full article

(This article belongs to the Section RNA)

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16 pages, 644 KB

Open AccessArticle

Forensic DNA Recovery from FFPE Tissue Using the Maxwell^® RSC Xcelerate Kit: Optimization, Challenges, and Limitations

by Dagmara Lisman, Andrzej Ossowski, Aleksandra Tołoczko-Grabarek, Mateusz Kozłowski and Aneta Cymbaluk-Płoska

Genes 2025, 16(9), 1074; https://doi.org/10.3390/genes16091074 - 12 Sep 2025

Abstract

Background/Objectives: Obtaining reliable DNA profiles from archival tissue preserved as formalin-fixed, paraffin-embedded (FFPE) samples remains a major challenge in both forensic and medical evaluations. The quality of DNA isolated from FFPE material is frequently compromised due to formalin-induced fragmentation and chemical modifications. These [...] Read more.

Background/Objectives: Obtaining reliable DNA profiles from archival tissue preserved as formalin-fixed, paraffin-embedded (FFPE) samples remains a major challenge in both forensic and medical evaluations. The quality of DNA isolated from FFPE material is frequently compromised due to formalin-induced fragmentation and chemical modifications. These limitations are particularly relevant in cases of suspected medical malpractice related to cancer diagnosis or treatment, where retrospective molecular analyses may provide critical evidence. The aim of this study was to evaluate the performance of the Maxwell^® RSC Xcelerate DNA FFPE Kit (Promega) in generating DNA profiles from archival FFPE tissue blocks of endometrial cancer and to identify the limitations associated with this approach. Methods: Archival FFPE blocks of endometrial cancer were analyzed using the Maxwell^® RSC Xcelerate DNA FFPE Kit. DNA yield, purity, and degradation indices were assessed using standard real-time PCR-based quantification methods. Short tandem repeat (STR) profiling was performed with forensic genotyping kits, and the completeness, allele balance, and reliability of obtained profiles were evaluated. The obtained results were compared with reference quality thresholds commonly used in forensic practice. Results: The Maxwell^® RSC Xcelerate Kit allowed for recovery of relatively high DNA yields with consistently low degradation indices, confirming good extraction efficiency from FFPE samples. Nevertheless, despite favorable quantitative values, the generation of complete STR profiles was often unsuccessful. Partial or incomplete profiles were frequent, characterized by allele dropout and imbalance, which substantially reduced their evidentiary value. These findings suggest that DNA fragmentation and fixation-related artifacts impair amplification efficiency and limit the usefulness of STR analysis. Conclusions: This study emphasizes the persistent challenges of DNA profiling from FFPE tissue in forensic-medical contexts. Although the Maxwell^® RSC Xcelerate Kit demonstrated effective DNA recovery, the ability to generate complete and interpretable STR profiles remained limited. Further refinement of extraction protocols, as well as improved interpretative strategies, are required to enhance the reliability and evidentiary significance of molecular analyses based on archival FFPE material. Full article

(This article belongs to the Special Issue Advanced Research in Forensic Genetics)

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10 pages, 1475 KB

Open AccessBrief Report

Rapid Documentation of Possible Semen Stains for Forensic DNA Profiling

by Zhonghui Thong, Audrey Ping Jue Wee, Baoqiang Heng and Christopher Kiu Choong Syn

Genes 2025, 16(9), 1073; https://doi.org/10.3390/genes16091073 - 12 Sep 2025

Abstract

The acid phosphatase (AP) test is widely utilised in forensic biology laboratories to examine for the presence of semen on crime evidence. If semen is present, the AP-positive areas are marked on the exhibit to indicate the precise location of the semen stain. [...] Read more.

The acid phosphatase (AP) test is widely utilised in forensic biology laboratories to examine for the presence of semen on crime evidence. If semen is present, the AP-positive areas are marked on the exhibit to indicate the precise location of the semen stain. However, documenting AP-positive areas with a crayon is time-consuming and laborious. In this proof-of-concept study, we evaluated the use of Saral Wax-Free Transfer Taper (TP) as an alternative tool for tracing the boundaries of AP-positive areas. We demonstrated that the TP pigment did not inhibit PCR amplification, as indicated by consistent internal PCR control (IPC) C_T values during real-time DNA quantification. While a reduction in DNA yield was observed under stress-test conditions, where TP pigment was intentionally included in the samples, complete STR profiles were still recovered with no allele dropout. Importantly, the documenting time for AP mapping was reduced by approximately five-fold with TP compared to crayon, underscoring its potential to enhance efficiency in forensic laboratory workflows. Full article

(This article belongs to the Section Molecular Genetics and Genomics)

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14 pages, 6055 KB

Open AccessArticle

Genome-Wide Identification of TPL/TPR Gene Family in Ten Cotton Species and Function Analysis of GhTPL3 Involved in Salt Stress Response

by Ganggang Zhang, Jianguo Gao, Faren Zhu, Kailu Chen, Jiliang Fan, Lu Meng, Zihan Li, Shandang Shi and Hongbin Li

Genes 2025, 16(9), 1072; https://doi.org/10.3390/genes16091072 - 12 Sep 2025

Abstract

Background/Objectives: The TOPLESS (TPL) and TOPLESS-related (TPR) proteins represent a highly conserved class of transcriptional co-repressors in plants, playing pivotal roles in modulating growth, development, and stress responses through the repression of key transcriptional regulators. However, a comprehensive genome-wide analysis of the TPL [...] Read more.

Background/Objectives: The TOPLESS (TPL) and TOPLESS-related (TPR) proteins represent a highly conserved class of transcriptional co-repressors in plants, playing pivotal roles in modulating growth, development, and stress responses through the repression of key transcriptional regulators. However, a comprehensive genome-wide analysis of the TPL/TPR gene family and its involvement in stress responses remains unexplored in cotton. Methods: In this study, 60 TPL/TPR genes were identified from the genomes of ten Gossypium species via bioinformatics approaches, and their protein physicochemical properties, gene structures, phylogenetic relationships, cis-regulatory elements, and expression profiles were characterized. Results: Chromosomal localization and collinearity analyses revealed that segmental duplication events have contributed to the expansion of the TPL/TPR gene family. Further examination of exon–intron architectures and conserved motifs highlighted strong evolutionary conservation within each TPL/TPR subgroup. Expression profiling demonstrated that TPL/TPR genes exhibit tissue-specific expression patterns, with particularly high transcript abundance in floral organs (e.g., petals and stigmas). Cis-element analysis suggested their potential involvement in multiple stress-responsive pathways. Notably, GhTPL3 showed high constitutive expression across various tissues and under stress conditions, with the most pronounced up-regulation under salt stress. Functional validation via Virus-Induced Gene Silencing (VIGS) confirmed that GhTPL3 silencing significantly impairs cotton salt stress tolerance, underscoring its critical role in abiotic stress adaptation. Conclusions: Our findings provide novel insights into the functional diversification and regulatory mechanisms of the TPL/TPR family in cotton, offering a valuable genetic resource for breeding stress-resilient cotton varieties. Full article

(This article belongs to the Special Issue Physiological and Molecular Mechanisms of Plant Stress Response)

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12 pages, 1929 KB

Open AccessArticle

Retinoic Acid-Regulated Epigenetic Marks Identify Alx1 as a Direct Target Gene Required for Optic Cup Formation

by Marie Berenguer and Gregg Duester

Genes 2025, 16(9), 1071; https://doi.org/10.3390/genes16091071 - 11 Sep 2025

Abstract

Background/Objectives: Retinoic acid (RA) is a transcriptional control agent that regulates several aspects of eye development including invagination of the optic vesicle to form the optic cup, although a target gene for this role has not been previously identified. As loss of RA [...] Read more.

Background/Objectives: Retinoic acid (RA) is a transcriptional control agent that regulates several aspects of eye development including invagination of the optic vesicle to form the optic cup, although a target gene for this role has not been previously identified. As loss of RA synthesis in Rdh10 knockout embryos affects the expression levels of thousands of genes, a different approach is needed to identify genes that are directly regulated by RA. Methods: Here, we combined ChIP-seq for the H3K27ac epigenetic mark with RNA-seq on optic field tissue from E10 wild-type and Rdh10−/− embryos that exhibit failure in optic cup formation. Results: We identified a small number of genes with decreased expression when RA is absent that also have a decreased presence of a nearby epigenetic gene activation mark (H3K27ac). One such gene was Alx1 that also has an RA response element (RARE) located near the RA-regulated H3K27ac mark, providing evidence that RA directly activates Alx1. In situ hybridization studies showed that Rdh10−/− embryos exhibit a large decrease of Alx1 expression in the optic field. CRISPR/Cas9 knockout of Alx1 resulted in a defect in optic cup formation due to a failure of perioptic mesenchyme to migrate and separate the optic cup epithelium from the forebrain neuroepithelium. Conclusions: Our studies support a model in which RA functions to directly activate Alx1 in perioptic mesenchyme to stimulate an early stage of eye development during which the optic vesicle folds into an optic cup that forms the retina. Full article

(This article belongs to the Special Issue Genetics and Genomics of Retinal Development and Diseases)

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Graphical abstract

30 pages, 18432 KB

Open AccessArticle

JCHAIN: A Prognostic Marker Based on Pan-Cancer Analysis to Inhibit Breast Cancer Progression

by Jinfeng Zhao, Wanquan Chen, Longpeng Li, Zhibin Zhang and Yaxin Wang

Genes 2025, 16(9), 1070; https://doi.org/10.3390/genes16091070 - 11 Sep 2025

Abstract

Background/Objectives: The JCHAIN (immunoglobulin-linked chain) is a multimeric IgA and IgM-linked chain whose involvement in oncogenesis and immunomodulation is unknown. The goal of this work was to conduct a comprehensive pan-cancer analysis of the JCHAIN to determine its expression profile, prognostic significance, immune [...] Read more.

Background/Objectives: The JCHAIN (immunoglobulin-linked chain) is a multimeric IgA and IgM-linked chain whose involvement in oncogenesis and immunomodulation is unknown. The goal of this work was to conduct a comprehensive pan-cancer analysis of the JCHAIN to determine its expression profile, prognostic significance, immune infiltration, and function in diverse malignancies. Methods: We performed pan-cancer analysis of gene expression data and protein expression data of JCHAIN using multiple databases, and analysed the prognostic significance of JCHAIN in a variety of cancers using univariate Cox analysis and Kaplan–Meier tools. The relationship between JCHAIN and immune cell infiltration was analysed via the TISIDB and TIMER websites, while single-cell and spatial transcriptomic analyses were performed to analyse the relationship between JCHAIN and the immune microenvironment. Mutations in the JCHAIN and their connection with methylation were then investigated using the cBioPortal and UALCAN websites. Afterwards, the function of JCHAIN was analysed by KEGG as well as GSEA, and the function of JCHAIN in breast cancer cells was verified by in vitro experiments. Results: The expression of the JCHAIN gene shows significant differences in most cancers, and its high expression is associated with a favourable prognosis. In most cancers, JCHAIN gene expression is closely linked to immune-related genes, immune cells, and methylation, as well as to being affected by mutations. In breast cancer, we found that the JCHAIN was negatively correlated with cellular stemness. Enrichment analysis indicated that the JCHAIN was involved in immune responses, B cell activation, and JAK-STAT signalling pathways. Functional experiments showed that overexpression of the JCHAIN inhibited tumour migration and invasion, which may be closely related to the activation of the IL-2/STAT4 signalling pathway. Conclusions: We found that JCHAIN can be used as a diagnostic and prognostic marker for a variety of cancers by pan-cancer analysis and verified that JCHAIN affects breast cancer cell progression through IL-2/STAT4 by in vitro experiments. Full article

(This article belongs to the Section Molecular Genetics and Genomics)

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9 pages, 941 KB

Open AccessCase Report

Mitochondrial Complex IV Deficiency Nuclear Type 11 Caused by a Novel Start-Lost Variant in the COX20 Gene

by Anna Kuchina, Artem Borovikov, Olga Sidorova, Maria Orlova, Oxana Ryzhkova, Igor Zaigrin and Aysylu Murtazina

Genes 2025, 16(9), 1069; https://doi.org/10.3390/genes16091069 - 11 Sep 2025

Abstract

Background: The COX20 gene encodes a critical assembly factor for cytochrome C oxidase (complex IV), and biallelic loss-of-function variants in this gene cause mitochondrial complex IV deficiency, typically presenting in infancy or childhood with hypotonia, ataxia, neuropathy, or dystonia. Methods: This study [...] Read more.

Background: The COX20 gene encodes a critical assembly factor for cytochrome C oxidase (complex IV), and biallelic loss-of-function variants in this gene cause mitochondrial complex IV deficiency, typically presenting in infancy or childhood with hypotonia, ataxia, neuropathy, or dystonia. Methods: This study describes an adult male patient with a broad clinical spectrum of central and peripheral nervous system involvement. Different medical genetic tests were performed for the patient, and only whole-genome trio sequencing identified pathogenic variants in the COX20 gene. A review of previously reported cases was conducted to compare clinical and imaging findings. Results: Two compound heterozygous COX20 variants in were identified: a known missense variant (c.41A>G; p.Lys14Arg) disrupting splicing, and a novel start-loss variant (c.2T>C; p.Met1?). The patient exhibited progressive ataxia, pyramidal signs, and peripheral neuropathy, accompanied by cervical spinal cord atrophy on spinal cord MRI and lower leg muscle fat infiltration on muscle MRI, an imaging feature not previously emphasized in COX20-related disease. Conclusions: A review of previously reported cases underscores broad clinical variability of the COX20-associated disorder, which may contribute to a prolonged diagnostic odyssey. Full article

(This article belongs to the Collection Genetics and Genomics of Rare Disorders)

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17 pages, 2726 KB

Open AccessArticle

Genome-Wide Association Study of Chlorophyll Fluorescence and Hyperspectral Indices in Drought-Stressed Young Plants in Maize

by Lovro Vukadinović, Vlatko Galić, Maja Mazur, Antun Jambrović and Domagoj Šimić

Genes 2025, 16(9), 1068; https://doi.org/10.3390/genes16091068 - 11 Sep 2025

Abstract

Background/Objectives: Global maize production is considerably affected by drought aggravated by climate change. No genome-wide association study (GWAS) or candidate gene analysis has been performed using chlorophyll fluorescence (ChlF) and hyperspectral (HS) indices measured in young plants challenged by a water deficit. Our [...] Read more.

Background/Objectives: Global maize production is considerably affected by drought aggravated by climate change. No genome-wide association study (GWAS) or candidate gene analysis has been performed using chlorophyll fluorescence (ChlF) and hyperspectral (HS) indices measured in young plants challenged by a water deficit. Our objective was to conduct a GWAS of nine ChlF and HS indices measured in a diversity panel of drought-stressed young plants grown in a controlled environment using a maize single nucleotide polymorphism (SNP) 50k chip. Methods: A total of 165 inbred lines were genotyped using the Infinium Maize50K SNP array and association mapping was carried out using a mixed linear model. Results: The GWAS detected 37 respective SNP markers significantly associated with the maximum quantum yield of the primary photochemistry of a dark-adapted leaf (Phi_Po), the probability that a trapped exciton moves an electron into the electron transport chain further than QA (Psi_o), the normalized difference vegetation index (NDVI), the Zarco–Tejada and Miller Index (ZMI), greenness, modified chlorophyll absorption in reflectance (MCARI), modified chlorophyll absorption in reflectance 1 (MCARI1), and Gitelson and Merzlyak indices 1 and 2 (GM1 and GM2). Conclusions: Our results contribute to a better understanding of the genetic dissection of the ChlF and HS indices, which is directly or indirectly related to physiological processes in maize, supporting the use of HS imaging in the context of maize breeding. Full article

(This article belongs to the Special Issue Molecular Breeding and Genetics of Plant Drought Resistance)

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16 pages, 1448 KB

Open AccessArticle

Exceptions to Broad Tissue-Specific Transcriptomic Interdependence: Searching for Independence in Expression of Genes

by Mikołaj Danielewski, Jarosław Walkowiak, Karolina Wielgus and Jan Krzysztof Nowak

Genes 2025, 16(9), 1067; https://doi.org/10.3390/genes16091067 - 10 Sep 2025

Abstract

Background: Correlation of genes within tissues has attracted much attention. In contrast, genes that are INDependent In Expression (INDIE) remain poorly understood, even though they may represent tissue admixtures, reflect new regulatory mechanisms, either transcriptional or post-transcriptional, and contribute to biomarkers or machine [...] Read more.

Background: Correlation of genes within tissues has attracted much attention. In contrast, genes that are INDependent In Expression (INDIE) remain poorly understood, even though they may represent tissue admixtures, reflect new regulatory mechanisms, either transcriptional or post-transcriptional, and contribute to biomarkers or machine learning algorithms. We hypothesised that INDIE genes can be found, may remain uncorrelated across tissues, and replicate within tissues in external datasets. Methods: Biweight midcorrelation was calculated for each gene against all other genes with sufficiently high expression in the given tissue from the GTEx dataset v8, along with the means of absolute values of obtained correlation coefficients. The threshold for gene designation as INDIE was both absolute (r) and relative (Z-score), while the threshold for external validation in the whole blood (four datasets) and the ileum (two datasets) was relative. Results: Only one gene, RPL13P12, was INDIE in all the analysed GTEx tissues, but it did not replicate in the external datasets. In contrast, HIST1H2AD and TMEM176B were not only INDIE in GTEx whole blood but also replicated in all four external datasets, despite their heterogeneity. Moreover, ACAT2 replicated in both external ileal datasets. The haemoglobin gene HBB belonged to most widespread INDIE genes in various GTEx tissues and was validated in an external ileal dataset, pointing towards the importance of tissue heterogeneity in bulk samples. Conclusions: A set of genes exhibiting independent expression patterns across various tissues of GTEx was described. Results for each tissue are made available. Even though many findings can be explained by tissue heterogeneity, some results point towards interesting mechanisms of gene expression regulation. Full article

(This article belongs to the Section Molecular Genetics and Genomics)

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12 pages, 229 KB

Open AccessArticle

Congenital Diaphragmatic Hernia and Joint Laxity: A Putative Link with Heritable Connective Tissue Disorders

by Alessandra Di Pede, Monia Magliozzi, Laura Valfré, Maria Lisa Dentici, Flaminia Pugnaloni, Viola Alesi, Andrea Conforti, Irma Capolupo, Annabella Braguglia, Andrea Dotta, Pietro Bagolan, Antonio Novelli and Maria Cristina Digilio

Genes 2025, 16(9), 1066; https://doi.org/10.3390/genes16091066 - 10 Sep 2025

Abstract

Background/Objectives: The etiology of congenital diaphragmatic hernia (CDH) remains unknown in over 50% of cases, although multiple heterogeneous causative defects have been identified. Emerging evidence suggests that specific genes and molecular pathways involved in connective tissue biology may contribute to CDH development. Associations [...] Read more.

Background/Objectives: The etiology of congenital diaphragmatic hernia (CDH) remains unknown in over 50% of cases, although multiple heterogeneous causative defects have been identified. Emerging evidence suggests that specific genes and molecular pathways involved in connective tissue biology may contribute to CDH development. Associations between CDH and connective tissue disorders have been reported, including cases in Marfan syndrome and a prevalence of CDH in 34% of patients with arterial tortuosity syndrome. Noticing joint laxity in several CDH patients, we aimed to investigate the presence of genetic variants linked to connective tissue disorders in this subgroup, focusing on patients enrolled in the follow-up program at Bambino Gesù Children’s Hospital. Methods: We selected patients diagnosed with CDH who also exhibited joint laxity based on a positive Beighton scale. These individuals underwent molecular analysis targeting genes known to be associated with heritable connective tissue disorders. Results: Genetic testing revealed variants in several genes across our patient series. These included mutations in FBN1, FBN2, ZNF469, VEGFA, NOTCH1, ELN, MCTP2, and SMAD6. In some cases, the variants were inherited paternally, while others appeared de novo. Most of these variants were classified as of unknown significance according to ACMG guidelines. Conclusions: (1) Several “variants of unknown significance” in different genes causative for connective tissue disorders have been detected in half of the present series of patients with CDH and joint laxity; (2) although the majority of the variants are classified accordingly to the ACMG as “variants of unknown significance”, a role of predisposition or susceptibility to CDH cannot be excluded; (3) a precise clinical evaluation for features of connective disorders should be recommended in the diagnostic workflow of patients with CDH. Full article

(This article belongs to the Section Human Genomics and Genetic Diseases)

18 pages, 1821 KB

Open AccessArticle

Transcriptomic Signatures of Mitochondrial Dysfunction in Autism: Integrated mRNA and microRNA Profiling

by Richard E. Frye, Zoe Hill, Shannon Rose, Sandra McCullough, Patricia A. Porter-Gill and Pritmohinder S. Gill

Genes 2025, 16(9), 1065; https://doi.org/10.3390/genes16091065 - 10 Sep 2025

Abstract

Background: Prior work established that about a third of ASD-derived LCLs show excessive mitochondrial respiration and stress vulnerability—features divergent from both controls and classical mitochondrial disease. This study explores how mRNA and microRNA (miRNA) expression profiles distinguish subtypes of autism spectrum disorder (ASD) [...] Read more.

Background: Prior work established that about a third of ASD-derived LCLs show excessive mitochondrial respiration and stress vulnerability—features divergent from both controls and classical mitochondrial disease. This study explores how mRNA and microRNA (miRNA) expression profiles distinguish subtypes of autism spectrum disorder (ASD) defined by mitochondrial function. Methods: Lymphoblastoid cell lines (LCLs) from boys with ASD were classified into two groups: those with abnormal (AD-A) and normal (AD-N) mitochondrial function. RNA-seq compared mRNA and miRNA expression differences. Results: 24 mRNA differentially expressed genes (DEGs) (14 downregulated, 10 upregulated in AD-N vs. AD-A) were identified, implicating processes such as mRNA processing, immune response, cancer biology, and crucially, mitochondrial and nuclear activities. Notably, genes such as DEPTOR (an mTOR modulator) were upregulated in AD-A, highlighting dysregulation in the mTOR pathway—a central regulator of cellular metabolism, protein synthesis, autophagy, and mitochondrial function. miRNA analysis revealed 18 differentially expressed miRNAs (DEMs) upregulated and one downregulated in AD-N compared to AD-A. Several miRNAs (including hsa-miR-1273h-3p, hsa-miR-197-3p, and hsa-miR-199a-5p) targeted both the differentially expressed genes and pathways previously linked to ASD, such as mTOR, Calmodulin Kinase II, and mitochondrial regulation. Enrichment analyses indicated involvement regulation of cell growth and division, gene expression, immune regulation and cellular stress as well as mTOR signaling. Conclusions: These molecular signatures support the idea that mitochondrial dysfunction in ASD is tied to specific disruptions in the mTOR and PI3K/AKT signaling axes, influencing cell growth, autophagy, oxidative stress handling, and neuronal metabolism. The findings highlight a miRNA-mRNA regulatory network that may underpin mitochondrial dysfunction and ASD heterogeneity, suggesting avenues for subtype-specific biomarkers and targeted therapies that address energy metabolism and cellular stress in ASD. Full article

(This article belongs to the Section Human Genomics and Genetic Diseases)

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14 pages, 3588 KB

Open AccessReview

Resistance Mutations in CLL: Genetic Mechanisms Shaping the Future of Targeted Therapy

by Samantha Sekeres, Erica N. Lamkin, Eduardo Bravo, Jr., Allison Cool and Justin Taylor

Genes 2025, 16(9), 1064; https://doi.org/10.3390/genes16091064 - 10 Sep 2025

Abstract

Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in Western populations and remains incurable despite significant therapeutic advancements. Over the past decade, the treatment landscape has evolved from traditional chemoimmunotherapy to targeted oral agents, including Bruton’s tyrosine kinase inhibitors (BTKis) [...] Read more.

Chronic lymphocytic leukemia (CLL) is the most common type of leukemia in Western populations and remains incurable despite significant therapeutic advancements. Over the past decade, the treatment landscape has evolved from traditional chemoimmunotherapy to targeted oral agents, including Bruton’s tyrosine kinase inhibitors (BTKis) and BCL2 inhibitors (BCL2is), which have demonstrated superior efficacy and tolerability, especially in elderly patients. Venetoclax, a BCL2i, induces apoptosis in CLL cells through selective inhibition of the anti-apoptotic BCL2 protein, while BTKis, such as ibrutinib and its next-generation analogs, disrupt B-cell receptor signaling critical to CLL cell survival. However, resistance to both drug classes has emerged, including mutations in BTK and BCL2, prompting the exploration of novel therapeutic strategies. This review outlines the molecular basis and clinical implications of these resistance mechanisms, as well as emerging therapeutic solutions, including non-covalent BTKis like pirtobrutinib and BTK-targeting PROTAC degraders such as BGB-16673 and NX-2127. Additionally, we discuss promising combination therapies incorporating BTKis, BCL2is, and anti-CD20 monoclonal antibodies. Finally, we highlight the growing role of measurable residual disease (MRD) as a biomarker to guide treatment duration and evaluate therapeutic success. As resistance mechanisms continue to emerge, tailoring therapy based on underlying biology will be critical to sustaining disease control and enhancing outcomes in patients with CLL. Full article

(This article belongs to the Special Issue Progress in Hematology: Non-Malignant, Pre-Malignant, and Malignant Disorders, and Genetically Based Therapies)

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18 pages, 2638 KB

Open AccessArticle

RNA Polymerase I Dysfunction Underlying Craniofacial Syndromes: Integrated Genetic Analysis Reveals Parallels to 22q11.2 Deletion Syndrome

by Spencer Silvey, Scott Lovell and Merlin G. Butler

Genes 2025, 16(9), 1063; https://doi.org/10.3390/genes16091063 - 10 Sep 2025

Abstract

Background/Objective: POLR1A and related gene variants cause craniofacial and developmental syndromes, including Acrofacial Dysostosis-Cincinnati, Treacher-Collins types 2–4, and TWIST1-associated disorders. Using a patient case integrated with molecular analyses, we aimed to clarify shared pathogenic mechanisms and propose these conditions as part of a [...] Read more.

Background/Objective: POLR1A and related gene variants cause craniofacial and developmental syndromes, including Acrofacial Dysostosis-Cincinnati, Treacher-Collins types 2–4, and TWIST1-associated disorders. Using a patient case integrated with molecular analyses, we aimed to clarify shared pathogenic mechanisms and propose these conditions as part of a spectrum of RNA polymerase I (Pol I)–related ribosomopathies. Methods: A patient with a heterozygous POLR1A variant underwent clinical evaluation. Findings were integrated with a literature review of craniofacial syndromes to identify overlapping fea tures. Protein-protein and gene-gene interactions were analyzed with STRING and Pathway Commons, a structural modeling of POLR1A assessed the mutation’s impact. Results: The patient exhibited features overlapping with Sweeney-Cox, Saethre-Cox, Robinow-Sorauf, and Treacher-Collins types 2–4, supporting a shared spectrum. Computational analyses identified POLR1A-associated partners and pathways converging on Pol I function, ribosomal biogenesis, and nucleolar processes. Structural modeling of the Met496Ile variant suggested disruption of DNA binding and polymerase activity, linking molecular dysfunction to the clinical phenotype. Conclusion: Significant clinical and genetic overlap exists among Saethre-Chotzen, Sweeney-Cox, Treacher-Collins types 2–4, and Acrofacial Dysostosis-Cincinnati. POLR1A and related Pol I subunits provide a mechanistic basis through impaired nucleolar organization and rRNA transcription, contributing to abnormal craniofacial development. Integrative protein, gene, and structural analyses support classifying these syndromes as Pol I–related ribosomopathies, with implications for diagnosis, counseling, and future mechanistic or therapeutic studies. Full article

(This article belongs to the Special Issue 15th Anniversary of Genes: Feature Papers in “Neurogenetics and Neurogenomics”)

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13 pages, 1571 KB

Open AccessArticle

CREPE (CREate Primers and Evaluate): A Computational Tool for Large-Scale Primer Design and Specificity Analysis

by Jonathan W. Pitsch, Sara A. Wirth, Nicole T. Costantino, Josh Mejia, Rose M. Doss, Ava V. A. Warren, Jack Ustanik, Xiaoxu Yang and Martin W. Breuss

Genes 2025, 16(9), 1062; https://doi.org/10.3390/genes16091062 - 10 Sep 2025

Abstract

Background/Objectives: Polymerase chain reaction (PCR) is ubiquitous in biological research labs, as it is a fast, flexible, and cost-effective technique to amplify a DNA region of interest. However, manual primer design can be an error-prone and time-consuming process depending on the number and [...] Read more.

Background/Objectives: Polymerase chain reaction (PCR) is ubiquitous in biological research labs, as it is a fast, flexible, and cost-effective technique to amplify a DNA region of interest. However, manual primer design can be an error-prone and time-consuming process depending on the number and composition of target sites. While Primer3 has emerged as an accessible tool to solve some of these issues, additional computational pipelines are required for appropriate scaling. Moreover, this does not replace the manual confirmation of primer specificity (i.e., the assessment of off-targets). Methods: To overcome the challenges of large-scale primer design, we fused the functionality of Primer3 and In-Silico PCR (ISPCR); this integrated pipeline, CREPE (CREate Primers and Evaluate), performs primer design and specificity analysis through a custom evaluation script for any given number of target sites at scale. Results: CREPE’s final output summarizes the lead forward and reverse primer pair for each target site, a measure of the likelihood of binding to off-targets, and additional information to aid decision-making. We provide this through a customized workflow for targeted amplicon sequencing (TAS) on a 150 bp paired-end Illumina platform. Experimental testing showed successful amplification for more than 90% of primers deemed acceptable by CREPE. Conclusions: We here provide CREPE, a software platform that allows for parallelized primer design for PCR applications and that is optimized for targeted amplicon sequencing. Full article

(This article belongs to the Section Bioinformatics)

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15 pages, 1327 KB

Open AccessArticle

PTEN Gene and Autism: Genetic Underpinnings and Neurodevelopmental Impacts

by Ann C. Genovese and Merlin G. Butler

Genes 2025, 16(9), 1061; https://doi.org/10.3390/genes16091061 - 9 Sep 2025

Abstract

Background/Objectives: Twin and family studies suggest that 90% of the risk for autism spectrum disorder (ASD) is due to genetic factors, with 800 genes recognized as playing a role. An important gene is phosphatase and tensin homolog (PTEN), which plays [...] Read more.

Background/Objectives: Twin and family studies suggest that 90% of the risk for autism spectrum disorder (ASD) is due to genetic factors, with 800 genes recognized as playing a role. An important gene is phosphatase and tensin homolog (PTEN), which plays a significant role in cancer as a tumor suppressor best known for causing overgrowth and PTEN hamartoma tumor syndromes (PHTS). Less well known are PTEN germline mutations with adverse neurodevelopmental impacts of macrocephaly, intellectual disability, and ASD, as well as other behavioral and psychiatric disturbances. There remains a limited understanding of whether these gene variants are associated with differing manifestations of PTEN-associated neurodevelopmental disorders. Methods: This review utilized comprehensive literature searches such as PubMed, OMIM, and Gene Reviews with keywords of PTEN, genetic factors, autism, and human studies and by searching genomic-protein functional networks with STRING computer-based programs for functional and genetic mechanisms. Results: This review explored the genetic underpinnings of PTEN gene variants causing altered interactive proteins and their mechanisms, biological processes, molecular functions, pathways, and disease–gene associations. We characterized specific gene–gene or protein–protein interactions and their functions relating to neurodevelopment, psychiatric disorders, and ASD that were found to be increased with PTEN gene variants. Conclusions: PTEN gene defects are among the most recognized genetic causes of ASD. PTEN gene variants and altered protein interactions and mechanisms described in our study are associated with an increased risk for tissue and organ overgrowth, macrocephaly, and distinct brain anomalies, specifically newly identified abnormal CSF dynamics. These genetic underpinnings and impacts on neurodevelopment are discussed. The genetic and protein findings identified may offer clues to effective treatment interventions, particularly when instituted at a young age, to improve long-term outcomes. Full article

(This article belongs to the Section Neurogenomics)

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18 pages, 604 KB

Open AccessReview

MicroRNA (miRNA) in the Pathogenesis of Diabetic Retinopathy: A Narrative Review

by Stamatios Lampsas, Chrysa Agapitou, Alexandros Chatzirallis, Georgios Papavasileiou, Dimitrios Poulakis, Sofia Pegka, Panagiotis Theodossiadis, Vaia Lambadiari and Irini Chatziralli

Genes 2025, 16(9), 1060; https://doi.org/10.3390/genes16091060 - 9 Sep 2025

Abstract

Diabetic retinopathy (DR) is the most common microvascular complication associated with diabetes mellitus and represents a leading cause of visual impairment worldwide. Inflammation, endothelial dysfunction, angiogenesis, neurodegeneration, and oxidative stress are key pathogenic processes in the development and progression of DR. Numerous microRNAs [...] Read more.

Diabetic retinopathy (DR) is the most common microvascular complication associated with diabetes mellitus and represents a leading cause of visual impairment worldwide. Inflammation, endothelial dysfunction, angiogenesis, neurodegeneration, and oxidative stress are key pathogenic processes in the development and progression of DR. Numerous microRNAs (miRNAs) show altered expression in DR and modulate critical biological pathways. Pro-inflammatory miRNAs such as miR-155 and miR-21 promote cytokine release and vascular inflammation, while miR-146a acts as a negative regulator of Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling. MiR-126 and miR-21 regulate endothelial integrity and angiogenesis through pathways involving Vascular Endothelial Growth Factor (VEGF). MiR-200b and miR-126 are downregulated in DR, leading to increased neovascularization via activation of the VEGF/ Mitogen-Activated Protein Kinase (MAPK) cascade. Apoptotic processes are affected by miR-195, which downregulates Sirtuin 1 (SIRT1) and B-cell lymphoma 2 (Bcl-2), promoting retinal cell death, while miR-29b downregulation permits upregulation of the transcription factor SP1, enhancing caspase-mediated apoptosis in Müller cells and endothelial cells. miRNAs collectively modulate an intricate regulatory network that contributes to the underlying mechanisms of diabetic retinopathy development and progression. This narrative review aims to summarize knowledge regarding the mechanisms miRNAs mediating pathogenetic mechanisms of DR. Full article

(This article belongs to the Section RNA)

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18 pages, 2156 KB

Open AccessArticle

Destructive and Non-Destructive Methods for aDNA Isolation from Teeth and Their Analysis: A Comparison

by Agnieszka Dobosz, Anna Jonkisz, Arleta Lebioda, Jerzy Kawecki and Tadeusz Dobosz

Genes 2025, 16(9), 1059; https://doi.org/10.3390/genes16091059 - 9 Sep 2025

Abstract

Background/Objectives: DNA analysis can be used to expand our understanding of extinct populations and the history of the world and humankind. Dental cavities often contain uncontaminated remains of ancient DNA (aDNA). Archaeological excavations are a convenient source for various samples; however, in almost [...] Read more.

Background/Objectives: DNA analysis can be used to expand our understanding of extinct populations and the history of the world and humankind. Dental cavities often contain uncontaminated remains of ancient DNA (aDNA). Archaeological excavations are a convenient source for various samples; however, in almost all extraction methods, a piece of bone or tooth is powdered before extraction, thereby causing damage to archaeological samples that are often irreplaceable and unique. This study aimed to develop a method that enables the collection of DNA from teeth without causing significant damage. Methods: This study presents two methods of DNA extraction from teeth: destructive and non-destructive. Both contemporary and archaeological teeth were examined using both destructive and non-destructive approaches to compare their efficiency. To verify the results, methods such as quantitative RT-PCR, STR analysis, and Y-SNP analysis were employed. Results: Extraction efficiency plays a critical role in this field of research. The main steps of the DNA extraction method were compared and optimized based on purification and using quantitative PCR. Conclusions: The results demonstrate that a non-destructive method of DNA isolation from human teeth can be used successfully, especially when teeth are unique and cannot be destroyed during the examination process. This method yields an appropriate amount of DNA for sequencing. Full article

(This article belongs to the Section Technologies and Resources for Genetics)

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10 pages, 219 KB

Open AccessReview

The Impact of Genetic Polymorphisms on the Clinical Efficacy of Azole Antifungals

by Hareesh Singam and Sherif Mossad

Genes 2025, 16(9), 1058; https://doi.org/10.3390/genes16091058 - 9 Sep 2025

Abstract

Azoles are the primary agents for antifungal activity in clinical medicine due to their broad-spectrum efficacy and favorable safety profiles compared to older agents. Triazoles, including fluconazole, itraconazole, voriconazole, posaconazole, and isavuconazole, have varied pharmacokinetic and pharmacodynamic properties. This is due to various [...] Read more.

Azoles are the primary agents for antifungal activity in clinical medicine due to their broad-spectrum efficacy and favorable safety profiles compared to older agents. Triazoles, including fluconazole, itraconazole, voriconazole, posaconazole, and isavuconazole, have varied pharmacokinetic and pharmacodynamic properties. This is due to various polymorphisms in hepatic enzymes, necessitating genotype-guided dosing and therapeutic drug monitoring (TDM) to optimize treatment outcomes. This review highlights the clinical relevance of pharmacogenomics in azole therapy, particularly the role of cytochrome P450 (CYP450) enzyme polymorphisms in influencing drug levels, efficacy, and toxicity. Understanding these genetic and metabolic factors is essential for personalized antifungal treatment strategies, improving patient safety and therapeutic outcomes. Full article

(This article belongs to the Special Issue Progress in Hematology: Non-Malignant, Pre-Malignant, and Malignant Disorders, and Genetically Based Therapies)

29 pages, 994 KB

Open AccessReview

Angiogenic microRNAs in Systemic Sclerosis: Insights into Microvascular Dysfunction and Therapeutic Implications

by Marta Rusek

Genes 2025, 16(9), 1057; https://doi.org/10.3390/genes16091057 - 9 Sep 2025

Abstract

Systemic sclerosis (SSc) is a complex connective tissue disease that affects the skin and internal organs and is characterized by immune dysregulation, progressive fibrosis, and microvascular dysfunction. Chronic tissue ischemia, accompanied by impaired angiogenesis, leads to the gradual loss of small vessels, resulting [...] Read more.

Systemic sclerosis (SSc) is a complex connective tissue disease that affects the skin and internal organs and is characterized by immune dysregulation, progressive fibrosis, and microvascular dysfunction. Chronic tissue ischemia, accompanied by impaired angiogenesis, leads to the gradual loss of small vessels, resulting in clinical complications, such as Raynaud’s phenomenon, digital ulcers, pulmonary arterial hypertension, and renal crisis. Emerging evidence highlights the crucial regulatory role of microRNAs (miRNAs) in vascular homeostasis through the modulation of key signaling pathways and endothelial cell activity. Dysregulated miRNAs influence fibroblast proliferation, inflammatory responses, and immune cell activity in SSc, contributing to disease progression. Current knowledge is still limited, highlighting the need for further research to elucidate the miRNAs network involved in the etiopathogenesis of SSc. The use of miRNA-based biomarkers is gaining tremendous attention for early diagnosis, risk stratification, classification, and the prediction of therapeutic responses. This review provides insights into angiogenesis-related miRNAs involved in SSc pathogenesis, discusses their relevance as biomarkers, and explores their promise as therapeutic targets. Advancing our knowledge of miRNAs-mediated regulatory networks may open new possibilities for personalized approaches to SSc management. Full article

(This article belongs to the Section RNA)

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