Genes

29 pages, 575 KiB

Open AccessReview

Hereditary Breast Cancer: Comprehensive Risk Assessment and Prevention Strategies

by Eliza Del Fiol Manna, Davide Serrano, Laura Cazzaniga, Sara Mannucci, Cristina Zanzottera, Francesca Fava, Gaetano Aurilio, Aliana Guerrieri-Gonzaga, Matilde Risti, Mariarosaria Calvello, Irene Feroce, Monica Marabelli, Cecilia Altemura, Lucio Bertario, Bernardo Bonanni and Matteo Lazzeroni

Genes 2025, 16(1), 82; https://doi.org/10.3390/genes16010082 - 13 Jan 2025

Viewed by 106

Abstract

Women carrying pathogenic/likely pathogenic (P/LP) variants in moderate- or high-penetrance genes have an increased risk of developing breast cancer. However, most P/LP variants associated with breast cancer risk show incomplete penetrance. Age, gender, family history, polygenic risk, lifestyle, reproductive, hormonal, and environmental factors [...] Read more.

Women carrying pathogenic/likely pathogenic (P/LP) variants in moderate- or high-penetrance genes have an increased risk of developing breast cancer. However, most P/LP variants associated with breast cancer risk show incomplete penetrance. Age, gender, family history, polygenic risk, lifestyle, reproductive, hormonal, and environmental factors can affect the expressivity and penetrance of the disease. However, there are gaps in translating how individual genomic variation affects phenotypic presentation. The expansion of criteria for genetic testing and the increasing utilization of comprehensive genetic panels may enhance the identification of individuals carrying P/LP variants linked to hereditary breast cancer. Individualized risk assessment could facilitate the implementation of personalized risk-reduction strategies for these individuals. Preventive interventions encompass lifestyle modifications, chemoprevention, enhanced surveillance through breast imaging, and risk-reducing surgeries. This review addresses the current literature’s inconsistencies and limitations, particularly regarding risk factors and the intensity of preventive strategies for women with P/LP variants in moderate- and high-penetrance genes. In addition, it synthesizes the latest evidence on risk assessment and primary and secondary prevention in women at high risk of breast cancer. Full article

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

14 pages, 3490 KiB

Open AccessArticle

Evaluation of Genetic Diversity and Identification of Cultivars in Spray-Type Chrysanthemum Based on SSR Markers

by Manjulatha Mekapogu, So-Hyeon Lim, Youn-Jung Choi, Su-Young Lee and Jae-A Jung

Genes 2025, 16(1), 81; https://doi.org/10.3390/genes16010081 - 13 Jan 2025

Viewed by 199

Abstract

Background/Objectives: Chrysanthemum (Chrysanthemum morifolium), a key ornamental and medicinal plant, presents challenges in cultivar identification due to high phenotypic similarity and environmental influences. This study assessed the genetic diversity and discrimination of 126 spray-type chrysanthemum cultivars. Methods: About twenty-three simple sequence [...] Read more.

Background/Objectives: Chrysanthemum (Chrysanthemum morifolium), a key ornamental and medicinal plant, presents challenges in cultivar identification due to high phenotypic similarity and environmental influences. This study assessed the genetic diversity and discrimination of 126 spray-type chrysanthemum cultivars. Methods: About twenty-three simple sequence repeat (SSR) markers were screened for the discrimination of 126 cultivars, among which six SSR markers showed polymorphic fragments. Results: Results showed high polymorphism across six markers, with an average of 3.8 alleles per locus and a mean polymorphism information content (PIC) of 0.52, indicating strong discriminatory efficiency. The average observed heterozygosity (Ho) was 0.72, reflecting significant genetic diversity within the cultivars. Cluster analysis using the unweighted pair group method with arithmetic mean (UPGMA) grouped the cultivars into seven clusters, correlating well with the PCA. Bayesian population structure analysis suggested two primary genetic subpopulations. Conclusions: These findings confirm SSR markers as an effective tool for the genetic characterization and precise discrimination of spray type chrysanthemum cultivars, offering significant applications in breeding, cultivar registration, and germplasm conservation. The SSR marker-based approach thus provides a reliable and efficient strategy to enhance the management and commercialization of diverse chrysanthemum germplasm collections. Full article

(This article belongs to the Special Issue Genetics and Breeding of Ornamental Plants)

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Spray type chrysanthemum cultivars representing various floral types and colors used in this study. (i) Yellow cultivars—Yellow Marble (Single (S)), Early Bird (Double (D)), Gold Rich (Anemone (A)), and Golden Pangpang (Pompon (P)); (ii) Pink cultivars—Glory Pink (S), Donna Pink (D), Pink Diamond (A), and Pink Bubble (P); (iii) Green Cultivars—Field Green (S), Windmill Green (D), Green Diamond (A), and Green Pangpang (P); (iv) Purple Cultivars—SACHRY8617 (S), Purple Cone (D), Disco Club (A), and Purple Pangpang (P); (v) Red cultivars—Red Marble (S), 10B1-173 (D), Bradford (A), and Rexy Red (P); and (vi) Orange Cultivars—Light Up (S), Orange Pangpang (D), Chopin Orange (A), and Orange Ball (P). Full article ">Figure 2
Representative images of (i) PCR amplicons of six SSR markers tested and separated on agarose gel; and (ii & iii) chromatograms of SSR_51 and SSR_16 in different samples by ABI genetic analyzer 3100xl. Full article ">Figure 3
Dendrogram illustrating the classification of 126 spray chrysanthemum cultivars, established based on a UPGMA analysis using SSR markers. Various clusters are shown on the right side of the dendrogram. The scale at the top is the Euclidean distance. Full article ">Figure 4
Plot depicting the principle component analysis (PCA) of 126 chrysanthemum cultivars based on SSR markers. Full article ">Figure 5
Graphical illustration of the assessment of the best subpopulation numbers according to the appropriate K value. (i) Mean of ∆K values representing 15 independent runs with K = 1 to K = 10 based on LnP(K) values. (ii) The mean of ∆K showed a peak at K = 2. Full article ">Figure 6
Genetic relatedness of individuals from 126 chrysanthemum cultivars, as analyzed by the STRUCTURE software (v4.3.2). Y-axis values represent membership coefficients to subpopulations, while X-axis values indicate the individual chrysanthemum cultivar codes of cultivars. Full article ">

16 pages, 3676 KiB

Open AccessArticle

Chromosome 4 Duplication Associated with Strabismus Leads to Gene Expression Changes in iPSC-Derived Cortical Neurons

by Mayra Martinez-Sanchez, William Skarnes, Ashish Jain, Sampath Vemula, Liang Sun, Shira Rockowitz and Mary C. Whitman

Genes 2025, 16(1), 80; https://doi.org/10.3390/genes16010080 - 12 Jan 2025

Viewed by 232

Abstract

Background/Objectives: Strabismus is the most common ocular disorder of childhood. Three rare, recurrent genetic duplications have been associated with both esotropia and exotropia, but the mechanisms by which they contribute to strabismus are unknown. This work aims to investigate the mechanisms of the [...] Read more.

Background/Objectives: Strabismus is the most common ocular disorder of childhood. Three rare, recurrent genetic duplications have been associated with both esotropia and exotropia, but the mechanisms by which they contribute to strabismus are unknown. This work aims to investigate the mechanisms of the smallest of the three, a 23 kb duplication on chromosome 4 (hg38|4:25,554,985-25,578,843). Methods: Using CRISPR and bridging oligos, we introduced the duplication into the Kolf2.1J iPSC line. We differentiated the parent line and the line with the duplication into cortical neurons using a three-dimensional differentiation protocol, and performed bulk RNASeq on neural progenitors (day 14) and differentiated neurons (day 63). Results: We successfully introduced the duplication into Kolf2.1J iPSCs by nucleofecting a bridging oligo for the newly formed junction along with cas9 ribonucleoparticles. We confirmed that the cells had a tandem duplication without inversion or deletion. The parent line and the line with the duplication both differentiated into neurons reliably. There were a total of 37 differentially expressed genes (DEGs) at day 63, 25 downregulated and 12 upregulated. There were 55 DEGs at day 14, 18 of which were also DEGs at day 63. The DEGs included a number of protocadherins, several genes involved in neuronal development, including SLITRK2, CSMD1, and VGF, and several genes of unknown function. Conclusions: A copy number variant (CNV) that confers risk for strabismus affects gene expression of several genes involved in neural development, highlighting that strabismus most likely results from abnormal neural development, and identifying several new genes and pathways for further research into the pathophysiology of strabismus. Full article

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

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Schematic representation of genome editing to create the chromosome 4 duplication. (A) Representation for designed guide RNAs, bridging oligos for the 5′ site (blue section is the adaptor sequence and green section targets the 5’ end of the duplicated region), new desired junction site (red section targets the 3’ end of the duplicated region and green section targets the 5’ end of the duplicated region), and 3′ site (red section targets the 3’ end of the duplicated region and pink corresponds to adaptor sequence). (B) Schematic diagram of possible binding of the 5′ oligo, 3′ oligo and desired new junction, after which a new DNA strand is synthesized. Figure created with <a href="http://BioRender.com" target="_blank">BioRender.com</a>. (C) Pooled genotyping via PCR using primers for the new anticipated junction using junction ssODN oligo alone or 5′, junction and 3′ oligos. (D) End point PCR from control individual (no chromosome 4 duplication or strabismus, Ctrl), positive control (strabismic patient with presence of chromosome 4 duplication, +Ctrl), and selected edited iPSC sub-line (iPSC) testing for 5′ junction (Junction 1), 3′ junction (Junction 2) (to ensure no inversion) and new junction for duplication. The duplication junction in the iPSC line is slightly smaller than in the positive control individual because the iPSC duplication is 50 bp smaller than the originally identified duplication due to limitations in the design of the guide RNAs. Full article ">Figure 2
Whole-genome sequences from the new line with chromosome 4 duplication and parental line. (A) Read counts in chromosome 4, region of duplication in white bar, with a gain of read depth in the new line (red) compared with the parental line (blue). (B) Integrated Genome Viewer (IGV) views of sequencing reads from the chromosome 4 region of duplication (blue bar) from the new line (top and zoomed in below) and parental line (middle). In addition to the increased read depth, split reads are evident at the junctions (zoomed in below) that map to the newly created duplication junction. Full article ">Figure 3
Schematic differentiation and validation of generated cortical neurons. (A) Schematic overview of the cortical neuron differentiation method, from day 0 to day 63. Figure created with <a href="http://BioRender.com" target="_blank">BioRender.com</a>. (B) Cortical neurons were dissociated at day 63 and immunocytochemical staining for MAP2 was performed 14 days after dissociation, in the control line (Kolf2.1J) and cells with chromosome 4 duplication (duplicated). (C) Quantification of MAP2 positive soma in control line (Kolf2.1J) and cells with duplication (duplicated); t-test was performed. (D) Quality control clustering heatmap from iPSCs, neuroprogenitor cells and cortical neurons at days 0 (three replicates), 14 and 63, respectively (four replicates), for control cells (K) and cells with duplication (D). (E) Heatmap for stem cell marker and neuronal marker expression using log2(FPKM + 1) values, comparing iPSCs (day 0), neuroprogenitor cells (day 14) and differentiated cortical neurons (day 63). Full article ">Figure 4
Differentially expressed genes in cells with the duplication. (A,B) Volcano plots showing the significant DEGs between control cells and cells with the duplication at the neuroprogenitor stage (A) and fully mature cortical neurons (B). Blue points represent downregulated genes, and red points represent upregulated genes. (C) DiVenn plot showing the unique and common DEGs for NPCs and cortical neurons. (D) Heatmap showing the expressions (z-scores) of shared DEGs at NPCs and cortical neuron stage from four replicates of control cells and cells with duplication. Full article ">Figure 5
Heatmap of the DEGs in neuroprogenitors (A) and cortical neurons (B) and validation by qPCR (C). (A) Heatmap showing the expressions (z-scores) of differentially expressed genes in neuroprogenitor cells between controls and cells with duplication (fourth independent differentiations). (B) Heatmap showing the expressions (z-scores) of differentially expressed genes in cortical neurons between controls and cells with duplication, a pink brain next to the gene name represents a neuronal/synapse related function and a blue brain next to the gene name represents genes associated with neurodevelopmental disorders. (C) qRT-PCR validation from randomly selected upregulated and downregulated genes; total RNA from genotypes at day 14 and day 63 was used to analyze expression levels, and data were normalized using TBP as the reference gene. Full article ">Figure 6
Protein–protein interaction (PPI) network of the DEGs in cortical neurons. The PPI interaction network of 9 relevant mapped neuronal function-related DEGs at day 63, constructed in the STRING database (DEGs highlighted in yellow circles). Full article ">

17 pages, 945 KiB

Open AccessReview

Research Progress on Gene Regulation of Plant Floral Organogenesis

by Lixia Zhou, Amjad Iqbal, Mengdi Yang and Yaodong Yang

Genes 2025, 16(1), 79; https://doi.org/10.3390/genes16010079 - 12 Jan 2025

Viewed by 196

Abstract

Flowers, serving as the reproductive structures of angiosperms, perform an integral role in plant biology and are fundamental to understanding plant evolution and taxonomy. The growth and organogenesis of flowers are driven by numerous factors, such as external environmental conditions and internal physiological [...] Read more.

Flowers, serving as the reproductive structures of angiosperms, perform an integral role in plant biology and are fundamental to understanding plant evolution and taxonomy. The growth and organogenesis of flowers are driven by numerous factors, such as external environmental conditions and internal physiological processes, resulting in diverse traits across species or even within the same species. Among these factors, genes play a central role, governing the entire developmental process. The regulation of floral genesis by these genes has become a significant focus of research. In the AE model of floral development, the five structural whorls (calyx, corolla, stamens, pistils, and ovules) are controlled by five groups of genes: A, B, C, D, and E. These genes interact to give rise to a complex control system that governs the floral organsgenesis. The activation or suppression of specific gene categories results in structural modifications to floral organs, with variations observed across different species. The present article examines the regulatory roles of key genes, including genes within the MADS-box and AP2/ERF gene clusters, such as AP1, AP2, AP3, AG, STK, SHP, SEP, PI, and AGL6, as well as other genes, like NAP, SPL, TGA, PAN, and WOX, in shaping floral organ genesis. In addition, it analyzes the molecular-level effects of these genes on floral organ formation. The findings offer a deeper understanding of the genetic governance of floral organ genesis across plant species. Full article

(This article belongs to the Special Issue Forest Genetics and Plant Physiology)

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11 pages, 315 KiB

Open AccessArticle

The Impact of Storage Conditions on DNA Preservation in Human Skeletal Remains: A Comparison of Freshly Excavated Samples and Those Stored for 12 Years in a Museum Depot

by Tonja Jeromelj, Tamara Leskovar and Irena Zupanič Pajnič

Genes 2025, 16(1), 78; https://doi.org/10.3390/genes16010078 - 11 Jan 2025

Viewed by 320

Abstract

Background: As the field of ancient DNA research continues to evolve and produce significant discoveries, it is important to address the crucial limitations it still faces. Under conducive conditions, DNA can persist for thousands of years within human skeletal remains, but, as excavation [...] Read more.

Background: As the field of ancient DNA research continues to evolve and produce significant discoveries, it is important to address the crucial limitations it still faces. Under conducive conditions, DNA can persist for thousands of years within human skeletal remains, but, as excavation occurs, the environment abruptly changes, often leading to the loss of DNA and valuable genetic information. Proper storage procedures are needed to mediate DNA degradation and maintain sample integrity. This study aimed to investigate the impact of long-term storage under unregulated temperatures and humidity conditions on DNA preservation in human skeletal remains. Methods: To achieve this, archaeological petrous bones were used for DNA recovery. The DNA yield and degree of DNA degradation were compared for samples originating from historically and geographically equivalent archaeological sites, which differed in times of excavation and, consequently, in storage durations and conditions. DNA yield and the degree of DNA degradation were determined using real time PCR. Results: A significant reduction in the DNA yield and a borderline significant increase in the degree of DNA degradation were detected for samples stored at unregulated conditions for approximately 12 years. Conclusions: Our results show the imperative need for adhering to scientific recommendations regarding the optimal temperature and humidity in the long-term storage of human skeletal material. Full article

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

12 pages, 509 KiB

Open AccessReview

ADAR Therapeutics as a New Tool for Personalized Medicine

by Matteo Bertoli, Luca La Via and Alessandro Barbon

Genes 2025, 16(1), 77; https://doi.org/10.3390/genes16010077 - 11 Jan 2025

Viewed by 378

Abstract

In the field of RNA therapy, innovative approaches based on adenosine deaminases acting on RNA (ADAR)-mediated site-directed RNA editing (SDRE) have been established, providing an exciting opportunity for RNA therapeutics. ADAR1 and ADAR2 enzymes are accountable for the predominant form of RNA editing [...] Read more.

In the field of RNA therapy, innovative approaches based on adenosine deaminases acting on RNA (ADAR)-mediated site-directed RNA editing (SDRE) have been established, providing an exciting opportunity for RNA therapeutics. ADAR1 and ADAR2 enzymes are accountable for the predominant form of RNA editing in humans, which involves the hydrolytic deamination of adenosine (A) to inosine (I). This inosine is subsequently interpreted as guanosine (G) by the translational and splicing machinery because of their structural similarity. Intriguingly, the novel SDRE system leverages this recoding ability of ADAR proteins to correct the pathogenic G to A nucleotide mutations through a short, engineered guide RNA (gRNA). Thus, ADAR-mediated SDRE is emerging as a powerful tool to manipulate the genetic information at the RNA level and correct disease-causing mutations without causing damage to the genome. Further it is emerging as a new instrument for personalized medicine, since treatments can be tailored to the unique genetic mutations present in an individual patient. In this short review, we aimed to described the main approached bases on ADARs activity, highlighting their advantages and disadvantages. Full article

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

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11 pages, 4814 KiB

Open AccessCase Report

Compound Heterozygous p.(R124C) (Classic Lattice Corneal Dystrophy) and p.(R124H) (Granular Corneal Dystrophy Type 2) in TGFBI: Phenotype, Genotype, and Treatment

by Ji Sang Min, Tae-im Kim, Ikhyun Jun, R. Doyle Stulting, Changrae Rho, Sang Beom Han, Heeyoung Kim, Jinseok Choi, Jinu Han and Eung Kweon Kim

Genes 2025, 16(1), 76; https://doi.org/10.3390/genes16010076 - 11 Jan 2025

Viewed by 255

Abstract

(1) Background: The phenotypes of classic lattice corneal dystrophy (LCD) and granular corneal dystrophy type 2 (GCD2) that result from abnormalities in transforming growth factor β-induced gene (TGFBI) have previously been described. The phenotype of compound heterozygous classic LCD and GCD2, [...] Read more.

(1) Background: The phenotypes of classic lattice corneal dystrophy (LCD) and granular corneal dystrophy type 2 (GCD2) that result from abnormalities in transforming growth factor β-induced gene (TGFBI) have previously been described. The phenotype of compound heterozygous classic LCD and GCD2, however, has not yet been reported. (2) Case report: A 39-year-old male (proband) presented to our clinic complaining of decreased vision bilaterally. A slit-lamp examination revealed corneal opacities consistent with classic LCD. Contrast sensitivity (CS) was decreased. A genetic analysis performed with commercially available real-time polymerase chain reaction (PCR) showed both homozygous classic LCD and homozygous GCD2. Sanger sequencing performed in our lab suggested compound heterozygosity for c.370C>T and c.371G>A variants, which was confirmed by the TA cloning of exon 4 of TGFBI and sequencing of clones. Phototherapeutic keratectomy (PTK) was performed on the right eye of the proband, and the CS improved. (3) Conclusions: Compound heterozygous classic LCD and GCD2 produces clinical findings like that of severe, classic LCD. PTK can improve VA and CS, delaying the need for keratoplasty. Full article

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

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Slit-lamp photographs and Fourier-domain anterior segment optical coherence tomographs (FD-OCT) of the proband. Slit-lamp photographs of the right eye (A,B) and left eye (D,E) are also shown. Diffuse grayish-white opacities in the subepithelial and superficial stroma ((A,D); black arrows) and distinct refractile lattice lines spreading to the periphery ((B,E); magnified views are shown at the upper right) were observed in both corneas of the proband. FD-OCT images of the right eye (C) and left eye (F) are shown. Diffuse grayish-white opacities in the subepithelial and superficial stromal area (black arrowheads) and many distinct refractile lattice lines spreading to the periphery (green arrowheads) were observed. Full article ">Figure 2
Slit-lamp photographs of the right eye (A) and left eye (B) of the proband are shown. Many distinct refractile lattice lines spreading to the periphery near to the limbus (green arrowheads) were observed (A). Dense, thick lattice lines are seen in the mid-peripheral cornea (B). Slit-lamp photographs using retro-illumination of the right eye (C) and left eye (D) show 35 lines in the temporal half OD and 33 in the nasal half OS. Many dot-shaped opacities (40 OD and 23 OS) under retro-illumination (red arrows) and distinct refractile lattice lines spreading to the periphery (green arrowheads) were observed in both corneas of the proband (magnified views are shown at the right upper corners). Full article ">Figure 3
Slit-lamp photographs and Fourier-domain anterior segment optical coherence tomographic (FD-OCT) images of genetically confirmed classic LCD heterozygotes ((A,D,G); 32-year-old) ((B,E,H); 43-year-old) ((C,F,I); 40-year-old). Diffuse grayish-white opacities in the subepithelial and superficial stromal area (black arrows) are shown (A–C). Distinct refractile lattice lines (green arrowheads), which are less confluent (15, 12, and 14 lines in each half cornea of (D–F)) and thinner than those of the proband, spreading to the periphery are observed (D–F). The dot-shaped opacities are less numerous than those of the proband (13, 9, and 5 dots in each (D–F)) (red arrows). FD-OCT images of heterozygotes show fewer lattice lines (green arrowheads), while diffuse grayish-white opacities in the subepithelial and superficial stromal area (black arrows) were observed at various depths (G–I). Full article ">Figure 4
Slit-lamp photographs of other genetically confirmed GCD2 heterozygotes. Granular deposits (yellow arrows) and linear deposits (red arrowhead) were observed in the corneas of 40-year-old (A), 38-year-old (B), and 37-year-old heterozygotes (C). Full article ">Figure 5
Genetic analysis results of TGFBI in the proband (A,C) and proband’s mother (B,D). The nucleotides changed from normal sequence are shown in red highlight (A,B). The automatic reader reported a change of arginine to tyrosine in the proband (A) and a change of arginine to cysteine in the proband’s mother (B). Partial nucleotide sequences of exon 4 of the TGFBI gene of the proband shows both C and T curves at 370 nucleotide (red star) and both C and A curves at 371 nucleotide (blue star) (C). Partial nucleotide sequences of exon 4 of the TGFBI gene of the proband’s mother shows both C and T curves at 370 nucleotide (green star) (D). Full article ">Figure 6
Slit-lamp photographs of the proband’s right eye before PTK (A) and after PTK of 30 μm and additional 10 μm ablations (B,C) performed sequentially on the same day. Since the surgeon could not determine the depth of opacities precisely before PTK, an additional 10 μm ablation was performed with a slit-lamp examination after the 30 µm PTK to remove opacities and still preserve as much of the posterior stroma as possible for future additional PTKs. Full article ">Figure 7
Slit-lamp photographs (OD) of the proband 1 month (A) and 6 months (B) after PTK. FD-OCT images taken 1 month after treatment show that superficial opaque deposits in the superior half were removed while some opacities remained in the inferior half of the right eye inside the white dotted oval line ((C), OD). Intact superficial opaque deposits remained inside the white oval line in the left eye, where PTK was not performed ((D), OS). The ‘# of averages’ in the FD-OCT photo refers to the number of repeated scans and automatic averaging performed by the machine as part of its noise-reduction function. The photopic contrast sensitivity testing of the right, the treated eye, of the proband 1 month after PTK showed normal values at 1.5 cycles/degree and slightly low values at other frequencies (E). In contrast, the left eye showed very low contrast sensitivity at all spatial frequencies (F). Full article ">

19 pages, 6027 KiB

Open AccessArticle

The X-Linked Tumor Suppressor TSPX Regulates Genes Involved in the EGFR Signaling Pathway and Cell Viability to Suppress Lung Adenocarcinoma

by Tatsuo Kido, Hui Kong and Yun-Fai Chris Lau

Genes 2025, 16(1), 75; https://doi.org/10.3390/genes16010075 - 11 Jan 2025

Viewed by 251

Abstract

Background: TSPX is an X-linked tumor suppressor that was initially identified in non-small cell lung cancer (NSCLC) cell lines. However, its expression patterns and downstream mechanisms in NSCLC remain unclear. This study aims to investigate the functions of TSPX in NSCLC by identifying [...] Read more.

Background: TSPX is an X-linked tumor suppressor that was initially identified in non-small cell lung cancer (NSCLC) cell lines. However, its expression patterns and downstream mechanisms in NSCLC remain unclear. This study aims to investigate the functions of TSPX in NSCLC by identifying its potential downstream targets and their correlation with clinical outcomes. Methods: RNA-seq transcriptome and pathway enrichment analyses were conducted on the TSPX-overexpressing NSCLC cell lines, A549 and SK-MES-1, originating from lung adenocarcinoma and squamous cell carcinoma subtypes, respectively. In addition, comparative analyses were performed using the data from clinical NSCLC specimens (515 lung adenocarcinomas and 502 lung squamous cell carcinomas) in the Cancer Genome Atlas (TCGA) database. Results: TCGA data analysis revealed significant downregulation of TSPX in NSCLC tumors compared to adjacent non-cancerous tissues (Wilcoxon matched pairs signed rank test p < 0.0001). Notably, the TSPX expression levels were inversely correlated with the cancer stage, and higher TSPX levels were associated with better clinical outcomes and improved survival in lung adenocarcinoma, a subtype of NSCLC (median survival extended by 510 days; log-rank test, p = 0.0025). RNA-seq analysis of the TSPX-overexpressing NSCLC cell lines revealed that TSPX regulates various genes involved in the cancer-related signaling pathways and cell viability, consistent with the suppression of cell proliferation in cell culture assays. Notably, various potential downstream targets of TSPX that correlated with patient survival (log-rank test, p = 0.016 to 4.3 × 10⁻¹⁰) were identified, including EGFR pathway-related genes AREG, EREG, FOSL1, and MYC, which were downregulated. Conclusions: Our results suggest that TSPX plays a critical role in suppressing NSCLC progression by downregulating pro-oncogenic genes, particularly those in the EGFR signaling pathway, and upregulating the tumor suppressors, especially in lung adenocarcinoma. These findings suggest that TSPX is a potential biomarker and therapeutic target for NSCLC management. Full article

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

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TSPX expression levels in relation to the pathologic stage and survival ratios of lung adenocarcinoma and squamous cell carcinoma patients. (A) TSPX expression levels in 58 lung adenocarcinoma tumor (T)/non-tumor (NT) paired samples from TCGA. Expression values (RSEM normalized count values) were plotted, with paired samples linked by a solid line; blue, decrease; red, increase. The p-value of the Wilcoxon matched pairs signed rank test is shown. (B) TSPX expression levels in 59 NT and 515 lung adenocarcinoma cases. The latter were divided into the TSPX-high group (highest 25%, n = 129), TSPX-low (lowest 25% cases, n = 129), and TSPX-mid (n = 257) group. (C) Distributions of pathologic stages (I-IV) across the TSPX-low, TSPX-mid, and TSPX-high groups. Chi-squared test p-value is indicated. (D) Survival curves for the TSPX-high (red), TSPX-mid (brown), and TSPX-low (blue) groups. Log-rank test p-value is indicated. (E) TSPX expression levels in 51 lung squamous cell carcinoma tumor/non-tumor paired samples from TCGA, similar to A. (F) TSPX expression levels in 51 NT and 502 lung squamous cell carcinoma cases, categorized into TSPX-high (highest 25% cases, n = 126), TSPX-low (lowest 25% cases, n = 126), and TSPX-mid (n = 246) groups. (G) Distributions of pathologic stages between the TSPX-low, TSPX-mid, and TSPX-high groups for lung squamous cell carcinoma, similar to C. Red indicates Stage-IV. Chi-squared test p-value is indicated. (H) Survival curves for the TSPX-high (red), TSPX-mid (brown), and TSPX-low (blue) groups in lung squamous cell carcinoma. Log-rank test p-value is indicated. Full article ">Figure 2
Overexpression of TSPX inhibits cell proliferation and causes cell death in A549 cells. (A) Diagram of the tet-ON transgene activation system. Addition of doxycycline (Dox) in the culture medium recruited the transactivator rtTA onto the tetracycline responsive (TRE) promoter and activated the target transgene. (B) The expressions of EGFP and TSPX in the respective transduced A549 cells with (+) and without (−) Dox induction were confirmed by Western blot, using β-actin as a reference. (C) Immunofluorescence of EGFP (green, left), TSPX (red, middle), and DAPI staining (blue) in the respective transduced A549 cells at 24 h after Dox induction. Far right panels show the merged images of TSPX, EGFP, and DAPI staining. (D) Scratch tests for A549-tetON-EGFP cells (left) and A549-tetON-TSPX cells (right) under a Dox-induction condition. Far right panel shows a magnified image of the boxed area. Yellow line indicates the wound edges at 0 h. Pink arrows indicate detached A549-tetON-TSPX cells. (E) Annexin-V binding assay (red) at 48 h after Dox induction showed that the detached A549-tetON-TSPX cells were positively stained by Annexin-V conjugated with Alexa Fluor 594 (red), corresponding to dead or apoptotic cells. Yellow arrows indicate cells stained with Annexin-V/Alexa Fluor 594. (F) Cell proliferation of A549-tetON-TSPX cells was inhibited under the Dox-induction condition (+Dox, right) but not under uninduced condition (−Dox, left), comparing with A549-tetON-EGFP cells, indicating that the TSPX overexpression inhibited cell proliferation. Asterisks indicate the significant difference at Student’s t-test p-value < 0.05. Scale bar = 50 µm in (C), 100 µm in (D,E). Full article ">Figure 3
Overexpression of TSPX inhibits cell proliferation in SK-MES-1 cells. (A) The expressions of EGFP and TSPX in the respective transduced SK-MES-1 cells with (+) and without (−) Dox induction were confirmed by Western blot. β-actin was analyzed as a reference. (B) Immunofluorescence of EGFP (green), TSPX (red), and DAPI staining (blue) in the respective transduced SK-MES-1 cells at 24 h after Dox-induction. Right panels show the merged images of TSPX and DAPI staining. (C) Cell proliferation of MES1-tetON-TSPX cells was inhibited under the Dox-induction condition (+Dox, right) but not under uninduced condition (−Dox, left), comparing with MES1-tetON-EGFP cells, indicating that the TSPX overexpression inhibited cell proliferation in SK-MES-1 cells. Asterisks indicate the significant difference at Student’s t-test p-value < 0.05. (D) Scratch tests for MES1-tetON-TSPX cells (top panels) and MES1-tetON-EGFP cells (bottom panels) under a Dox-induction condition. Phase contrast images show the representative gaps at 0 h and 48 h after scratch and Dox-induction. Far right panels show digitally magnified images of the boxed areas, respectively. The scratched area was completely healed by MES1-tetON-EGFP, but not by MES1-tetON-TSPX cells at 48 h. Scale bar = 100 µm in (B), 200 µm in (D). Full article ">Figure 4
Identification of the TSPX downstream genes and their biological processes in A549 and SK-MES-1 cells. (A) Volcano plots representing DEGs between TSPX-overexpressing and control cells in A549 (left) and SK-MES-1 (right, abbreviated as MES1) cell lines. The list of DEGs is shown in <a href="#app1-genes-16-00075" class="html-app">Supplementary Table S1</a>. (B) Results of the DAVID pathway enrichment analysis for DEGs in TSPX-overexpressing A549 and SK-MES-1 cells. The pathways that were commonly enriched in both A549 and SK-MES-1 cells are shown on the right. A magnified image of all enriched pathways, including the cell line-specific pathways, is shown in <a href="#app1-genes-16-00075" class="html-app">Supplementary Figure S1</a>. (C) Gene expression changes in DEGs involved in the selected pathways in (B). A549, DEGs in TSPX-overexpressing A549 cells; MES1, DEGs in TSPX-overexpressing SK-MES-1 cells. Only DEGs shared between A549 and SK-MES-1 cells are shown. Arrows indicate the genes analyzed in (D). (D) Validation of the gene expression changes induced by TSPX overexpression in A549 cells and SK-MES-1 cells by quantitative RT-PCR. Values represent the relative expression levels of AREG, DKK1, EREG, FOSL1, and MYC in A549-tetON-TSPX cells or MES1-tetON-TSPX cells (TSPX) at 24 h after Dox induction (mean ± standard error, n = 3). A549-tetON-EGFP cells and MES1-tetON-EGFP cells were used as references (EGFP), respectively. Values were normalized against GAPDH as an internal control. **, Student’s t-test p-value ≤ 0.001; ***, p-value ≤ 0.0001. Full article ">Figure 5
Gene expression patterns of TSPX-downstream genes in clinical lung adenocarcinoma from TCGA datasets. (A) Expression levels (RSEM normalized read counts) of AREG, BIRC3, CXCL5, DKK1, EREG, FOSL1, MYC, PLAU, and CACNA2D2, in non-tumor lung tissues (NT), TSPX-low lung adenocarcinoma group (TSPX-low), and TSPX-high lung adenocarcinoma group (TSPX-high) are shown. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparison test; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001; ns, not significant. Error bars represent mean ± SEM. (B) Correlation between the expression levels of the indicated genes and patient survival. Survival curves for high expressors (orange) or low expressors (green) are shown. Log-rank test p-values were obtained from TCGA datasets via the Human Protein Atlas (HPA). Full article ">Figure 6
Schematic diagram illustrating the functions of the identified TSPX-downstream genes in the EGFP-signaling and other pathways involved in cell survival in lung adenocarcinoma. Details of respective functions are described in the body text. Full article ">

14 pages, 1197 KiB

Open AccessReview

Hypertrophic Cardiomyopathy: New Clinical and Therapeutic Perspectives of an “Old” Genetic Myocardial Disease

by Chiara Calore, Mario Mangia, Cristina Basso, Domenico Corrado and Gaetano Thiene

Genes 2025, 16(1), 74; https://doi.org/10.3390/genes16010074 - 10 Jan 2025

Viewed by 298

Abstract

Since its first pathological description over 65 years ago, hypertrophic cardiomyopathy (HCM), with a worldwide prevalence of 1:500, has emerged as the most common genetically determined cardiac disease. Diagnostic work-up has dramatically improved over the last decades, from clinical suspicion and abnormal electrocardiographic [...] Read more.

Since its first pathological description over 65 years ago, hypertrophic cardiomyopathy (HCM), with a worldwide prevalence of 1:500, has emerged as the most common genetically determined cardiac disease. Diagnostic work-up has dramatically improved over the last decades, from clinical suspicion and abnormal electrocardiographic findings to hemodynamic studies, echocardiography, contrast-enhanced cardiac magnetic resonance, and genetic testing. The implementation of screening programs and the use of implantable cardioverter defibrillators (ICDs) for high-risk individuals have notably reduced arrhythmic sudden deaths, altering the disease’s mortality profile. Therapeutic breakthroughs, including surgical myectomy, alcohol septal ablation, and the novel introduction of “myosin inhibitors”, have revolutionized symptom management and reduced progression to advanced heart failure (HF) and death. Despite this progress, refractory HF—both with preserved and reduced systolic function—has become the predominant cause of HCM-related mortality. While most patients with HCM experience a favorable clinical course with low morbidity and mortality, timely identification and targeted treatment of high-risk subgroups progressing toward progressive HF remain a pressing challenge, even for expert clinicians. Full article

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

15 pages, 1390 KiB

Open AccessArticle

Population Genetics of Haliotis discus hannai in China Inferred Through EST-SSR Markers

by Hongsu Yang, Zhou Wu, Guangyu Ge, Xiujun Sun, Biao Wu, Zhihong Liu, Tao Yu, Yanxin Zheng and Liqing Zhou

Genes 2025, 16(1), 73; https://doi.org/10.3390/genes16010073 - 10 Jan 2025

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Abstract

Background/Objectives: The Pacific abalone Haliotis discus hannai originated in cold waters and is an economically important aquaculture shellfish in China. Our goal was to clarify the current status of the genetic structure of Pacific abalone in China. Methods: In this study, eighteen polymorphic [...] Read more.

Background/Objectives: The Pacific abalone Haliotis discus hannai originated in cold waters and is an economically important aquaculture shellfish in China. Our goal was to clarify the current status of the genetic structure of Pacific abalone in China. Methods: In this study, eighteen polymorphic EST-SSR loci were successfully developed based on the hemolymph transcriptome data of Pacific abalone, and thirteen highly polymorphic EST-SSR loci were selected for the genetic variation analysis of the six populations collected. Results: The results showed that the average number of observed alleles was 8.0769 (RC)-11.3848 (DQ) in each population. The number of observed alleles in the DQ, NH, and TJ populations was significantly higher than that in the RC population. The cultivated population outside the Changshan Islands has experienced a 22.79% reduction in allele diversity compared to the wild population of DQ. The pairwise F_st values and analysis of molecular variance (AMOVA) revealed significant population differentiation among all populations except DQ and NH populations, with RC and ZZ cultured populations exhibiting the largest population differentiation (F_st = 0.1334). The phylogenetic tree and structural analysis divided the six populations into two groups (group 1: NH, DQ, and ZZ; group 2: DL, TJ, and RC), and there was no relationship between geographical distance and genetic distance. Conclusions: These results may reflect the large-scale culture from different populations in China and the exchange of juveniles between hatcheries. Different breeding conditions have led to a higher degree of genetic differentiation between the RC and ZZ populations. This study enables a better understanding of the genetic diversity and structure of current Pacific abalone populations. Full article

(This article belongs to the Special Issue Genetic Studies of Fish)

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13 pages, 559 KiB

Open AccessReview

22q11.21 Deletions: A Review on the Interval Mediated by Low-Copy Repeats C and D

by Veronica Bertini, Francesca Cambi, Annalisa Legitimo, Giorgio Costagliola, Rita Consolini and Angelo Valetto

Genes 2025, 16(1), 72; https://doi.org/10.3390/genes16010072 - 9 Jan 2025

Viewed by 347

Abstract

22q11.2 is a region prone to chromosomal rearrangements due to the presence of eight large blocks of low-copy repeats (LCR22s). The 3 Mb 22q11.2 “typical deletion”, between LCR22-A and D, causes a fairly well-known clinical picture, while the effects of smaller CNVs harbored [...] Read more.

22q11.2 is a region prone to chromosomal rearrangements due to the presence of eight large blocks of low-copy repeats (LCR22s). The 3 Mb 22q11.2 “typical deletion”, between LCR22-A and D, causes a fairly well-known clinical picture, while the effects of smaller CNVs harbored in this interval are still to be fully elucidated. Nested deletions, flanked by LCR22B-D, LCR22B-C, or LCR22C-D, are very rare and are collectively described as “central deletions”. The LCR22C-D deletion (CDdel) has never been separately analyzed. In this paper, we focused only on CDdel, evaluating its gene content and reviewing the literature and public databases in order to obtain new insights for the classification of this CNV. At first glance, CDdels are associated with a broad phenotypic spectrum, ranging from clinically normal to quite severe phenotypes. However, the frequency of specific clinical traits highlights that renal/urinary tract abnormalities, cardiac defects, and neurological/behavioral disorders are much more common in CDdel than in the general population. This frequency is too high to be fortuitous, indicating that CDdel is a predisposing factor for these phenotypic traits. Among the genes present in this interval, CRKL is an excellent candidate for cardiac and renal defects. Even if further data are necessary to confirm the role of CDdels, according to our review, this CNV fits into the class of ‘likely pathogenic’ CNVs. Full article

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

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17 pages, 1057 KiB

Open AccessReview

Liquid Biopsy and Challenge of Assay Heterogeneity for Minimal Residual Disease Assessment in Colon Cancer Treatment

by Giovanni Crisafulli

Genes 2025, 16(1), 71; https://doi.org/10.3390/genes16010071 - 9 Jan 2025

Viewed by 393

Abstract

This review provides a comprehensive overview of the evolving role of minimal residual disease (MRD) for patients with Colon Cancer (CC). Currently, the standard of care for patients with non-metastatic CC is adjuvant chemotherapy (ACT) for all patients with stage III and high-risk [...] Read more.

This review provides a comprehensive overview of the evolving role of minimal residual disease (MRD) for patients with Colon Cancer (CC). Currently, the standard of care for patients with non-metastatic CC is adjuvant chemotherapy (ACT) for all patients with stage III and high-risk stage II CC following surgical intervention. Despite a 5–20% improvement in long-term survival outcomes, this approach also results in a significant proportion of patients receiving ACT without any therapeutic benefit and being unnecessarily exposed to the risks of secondary side effects. This underscores an unmet clinical need for more precise stratification to distinguish patients who necessitate ACT from those who can be treated with surgery alone. By employing liquid biopsy, it is possible to discern MRD enabling the categorization of patients as MRD-positive or MRD-negative, potentially revolutionizing the management of ACT. This review aimed to examine the heterogeneity of methodologies currently available for MRD detection, encompassing the state-of-the-art technologies, their respective advantages, limitations, and the technological challenges and multi-omic approaches that can be utilized to enhance assay performance. Furthermore, a discussion was held regarding the clinical trials that employ an MRD assay focusing on the heterogeneity of the assays used. These differences in methodology, target selection, and performance risk producing inconsistent results that may not solely reflect biological/clinical differences but may be the consequence of the preferential use of particular products in studies conducted in different countries. Standardization and harmonization of MRD assays will be crucial to ensure the liquid revolution delivers reliable and clinically actionable outcomes for patients. Full article

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

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17 pages, 5012 KiB

Open AccessArticle

Comprehensive Analysis of the NHX Gene Family and Its Regulation Under Salt and Drought Stress in Quinoa (Chenopodium quinoa Willd.)

by Yalla Santhoshi, Asha Bindhu Anjana, Harshvardhan Zala, Tejas Bosamia, Kapil Tiwari, Ketan Prajapati, Pranay Patel, Nishit Soni, Nitin Patel, Satyanarayan Solanki and Ulhas Sopanrao Kadam

Genes 2025, 16(1), 70; https://doi.org/10.3390/genes16010070 - 9 Jan 2025

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Abstract

Background/Objectives: Abiotic stresses such as salinity and drought significantly constrain crop cultivation and affect productivity. Quinoa (Chenopodium quinoa Willd.), a facultative halophyte, exhibits remarkable tolerance to drought and salinity stresses, making it a valued model for understanding stress adaptation mechanisms. The [...] Read more.

Background/Objectives: Abiotic stresses such as salinity and drought significantly constrain crop cultivation and affect productivity. Quinoa (Chenopodium quinoa Willd.), a facultative halophyte, exhibits remarkable tolerance to drought and salinity stresses, making it a valued model for understanding stress adaptation mechanisms. The objective of this study was to identify and characterize Sodium/Hydrogen antiporter (NHX) genes from the quinoa genome and study their role in stress tolerance. Methods: We identified and characterized 10 NHX genes from the quinoa genome, which belong to the monovalent cation/proton antiporter 1 (CPA1) superfamily. Comprehensive analysis, including phylogenetic relationships, motif patterns, and structural characteristics, was performed to classify these genes into three subfamilies. Physicochemical properties such as isoelectric point (pI), GRAVY, and transmembrane domains were examined. Promoter analysis was conducted to identify cis-elements linked to abiotic stress responses, phytohormone signalling, and light regulation. qPCR analysis was used to assess the differential expression patterns of CqNHX genes under salt and drought stress. Results: The analysis revealed that the NHX genes were divided into three subfamilies localized to vacuolar, plasma, and endosomal membranes. These genes exhibited structural and functional diversity. Promoter analysis indicated the presence of cis-elements associated with abiotic stress responses, phytohormone signalling, and light regulation, suggesting diverse regulatory roles. qPCR analysis revealed differential expression patterns of CqNHX genes under salt and drought stress, with vacuolar NHXs showing higher induction in leaf tissues under salinity. This underscores their critical role in sodium sequestration and ion homeostasis. Evolutionary analysis indicated a high degree of conservation within subfamilies, alongside evidence of purifying selection. Conclusions: The findings enhance our understanding of the molecular basis of stress tolerance in quinoa and provide valuable targets for genetic engineering to improve crop resilience to environmental challenges. Full article

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

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Phylogenetic relationship of NHX proteins from C. quinoa, A. thaliana (AtNHXs), Oryza sativa subsp. Indica (OsNHXs), Triticum aestivum (TaNHXs), Sorghum bicolor (SbNHXs), and Zea mays (ZmNHXs). The tree was constructed using the Neighbour-end joining method with 1000 bootstrap replicates. Full article ">Figure 2
Phylogenetic relation of the NHX proteins in quinoa and their conserved motif distribution. Proteins belonging to the same subfamily are enclosed in black boxes. The identified motifs are indicated by different colors. The sequence logos refer to 10 conserved motifs of CqNHXs. The conserved region “FFIYLLPPI” is represented by motif 3 (Red Box). The width of each motif (expressed in terms of amino acids) is given in the right-hand column. The height of the letters indicates the degree of conservation at that position. Less expectation (E) value explains higher confidence of prediction. The motifs are arranged according to their E value (low to high). Full article ">Figure 3
Multiple sequence alignment of amiloride-binding site (FFIYLLPPI) between quinoa NHXs and AtNHX1. The conserved sequence is separated by a box (red color). The region was present only in Vac-typed NHXs at their N-terminus. Exon and intron organization (gene structure) of CqNHXs was predicted by the GSDS webserver. The CDS (coding sequence) is represented by the color yellow, UTRs (untranslated regions) are shown by the color blue, and introns are indicated with solid black lines. The scale of gene lengths is given in kilo bases (kb) at the bottom. Full article ">Figure 4
Chromosomal distribution of CqNHX genes. The chromosome number is shown at the top and the position of NHX genes is indicated using solid black lines. On the right of each chromosome, the gene name is labelled. The scale of genome size is indicated in terms of mega bases (Mb) on the left. Ch0—unassembled chromosome. Full article ">Figure 5
Representation of cis-acting regulatory elements present in 1500 bp upstream of CqNHX promoters. The functional distribution of each cis-acting elements is presented at the top. The scale of abundance is shown on the right-hand side, with a dark red color and dark blue color indicating the higher and lower abundance of cis-acting elements, respectively. Full article ">Figure 6
Relative expression levels of CqNHX genes in leaves and roots of Chenopodium quinoa in response to salt stress for 0, 6, 12, and 24 h and drought stress for 0, 3, 5, and 7 days. The y-axis and x-axis represent the fold change and treatment periods, respectively. Error bars indicate standard error of CT means of NHX genes. Full article ">

13 pages, 5621 KiB

Open AccessArticle

Molecular Characterization, Recombinant Expression, and Functional Analysis of Carboxypeptidase B in Litopenaeus vannamei

by Hongmei Li, Hai Lin, Hao Yang, Chunhua Ren, Yi He, Xiao Jiang, Ting Chen and Chaoqun Hu

Genes 2025, 16(1), 69; https://doi.org/10.3390/genes16010069 - 9 Jan 2025

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Abstract

Background/Objectives: The Pacific white shrimp (L. vannamei) is economically significant, and its growth is regulated by multiple factors. Carboxypeptidase B (CPB) is related to protein digestion, but its gene sequence and features in L. vannamei are not fully understood. This study [...] Read more.

Background/Objectives: The Pacific white shrimp (L. vannamei) is economically significant, and its growth is regulated by multiple factors. Carboxypeptidase B (CPB) is related to protein digestion, but its gene sequence and features in L. vannamei are not fully understood. This study aimed to explore the molecular and functional properties of CPB in L. vannamei. Methods: The Lv-CPB gene was cloned, and bioinformatics analysis, qRT-PCR, in situ hybridization, recombinant protein expression in Escherichia coli, and an enzyme activity assay were performed. Results: The Lv-CPB gene is 1414 bp long with a 1263 bp ORF encoding a 420-amino-acid protein. It is stable, hydrophilic, and is highly expressed in the hepatopancreas. The recombinant protein was efficiently expressed with a molecular weight of about 47 kDa. The optimal pH and temperature for Lv-CPB were 8.0 and 50 °C, respectively. Conclusions: This study revealed the molecular and functional characteristics of Lv-CPB, providing insights into its role in shrimp digestion, as well as suggestions for improving aquaculture practices. Full article

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

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Full-length cDNA sequence and structural analysis of Lv-CPB. (A) The complete cDNA sequence of Lv-CPB and its corresponding translated amino acid sequence. The start codon ATG and stop codon TAG are highlighted in red. The signal peptide (SP) is shaded in blue, the activation peptide domain (APD) is marked in red, and the peptidase M14 carboxypeptidase domain (PMCD) is marked in green. A red box indicates the zinc ion-binding region, while the catalytic active site residues are enclosed in a yellow box. (B) Schematic representation of the structural domains of the Lv-CPB protein. (C) Schematic model of the Lv-CPB protein, with domains, zinc ion-binding site, and catalytic residues labeled as indicated. Full article ">Figure 2
Amino acid sequence alignment, phylogenetic, and 3D structure analysis of Lv-CPB. (A) Multiple sequence alignment of Lv-CPB with CPB from other species, where conserved amino acid residues are highlighted in darker shades, with the intensity indicating the degree of conservation. (B) Phylogenetic analysis of CPB across various species was conducted using the neighbor-joining method with a bootstrap value of 1000. (C) Structural superimposition of CPB protein models from closely related species, with sequences from different species represented in distinct colors. The PMCD is highlighted in red, and the APD is shown in black. Full article ">Figure 3
Tissue distribution and localization of Lv-CPB mRNA. (A) Expression profile of Lv-CPB mRNA across various tissues of shrimp, including TN (thoracic nerve), VN (ventral nerve cord), Ts (testis), Ov (ovary), Es (esophagus), Ht (heart), Gi (gill), Ms (muscle), In (intestine), St (stomach), Br (brain), He (hemolymph), and Hp (hepatopancreas). Data are presented as mean ± SE from three biological replicates. (B) Localization of Lv-CPB mRNA-positive cells in the hepatopancreas of L. vannamei. Hematoxylin and eosin (H&E) staining was performed on tissue sections. The negative control for ISH was conducted without a DIG-labeled DNA template. Lv-CPB mRNA-positive cells are distributed throughout the hepatopancreas. Full article ">Figure 4
Recombinant expression of the Lv-CPB protein. (A) Codon optimization, with original bases highlighted in blue and optimized bases in green. (B) Comparison of Lv-CPB protein expression levels in E. coli before and after codon optimization, with protein quantified in the soluble supernatant (SU) and inclusion fraction (IF). Arrows indicate target proteins (M: marker). (C) Comparison of expression efficiency under different concentrations of IPTG induction. “BK” represents the non-induced control group, “Low” corresponds to induction with 0.2 mM IPTG, and “High” corresponds to induction with 1.0 mM IPTG. (D) Comparison of Lv-CPB protein before and after purification, with PU representing the purified sample. Full article ">Figure 5
Enzymatic properties of Lv-CPB. (A) Standard curve of Lv-CPB’s enzymatic activity, where OD254 indicates the concentration of the catalytic product. (B) Activity plot of Lv-CPB as a function of pH. (C) Activity plot of Lv-CPB as a function of temperature. Full article ">

19 pages, 2164 KiB

Open AccessArticle

Chromosomal rDNA Distribution Patterns in Clonal Cobitis Triploid Hybrids (Teleostei, Cobitidae): Insights into Parental Genomic Contributions

by Alicja Boroń, Anna Grabowska, Olga Jablonska, Lech Kirtiklis, Sara Duda and Dorota Juchno

Genes 2025, 16(1), 68; https://doi.org/10.3390/genes16010068 - 9 Jan 2025

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Abstract

Background: Interspecific hybridization between relative species Cobitis taenia (with a diploid genome designated as TT), Cobitis elongatoides (EE) and Cobitis tanaitica (NN) and the successive polyploidization with transitions from sexuality to asexuality experienced by triploid Cobitis hybrids likely influence their chromosomal rearrangements, including [...] Read more.

Background: Interspecific hybridization between relative species Cobitis taenia (with a diploid genome designated as TT), Cobitis elongatoides (EE) and Cobitis tanaitica (NN) and the successive polyploidization with transitions from sexuality to asexuality experienced by triploid Cobitis hybrids likely influence their chromosomal rearrangements, including rearrangements of ribosomal DNA (rDNA) distribution patterns. Previously, we documented distinct karyotypic differences: C. elongatoides exhibited bi-armed chromosomes while C. taenia showed uni-armed chromosomes with rDNA-positive hybridization signals, respectively. Methods: In this study, fluorescence in situ hybridization (FISH) with 5S rDNA and 28S rDNA probes was used to analyze and compare chromosomal distribution patterns of rDNAs in clonally reproduced triploid Cobitis hybrids of different genomic constitutions ETT, ETN, EEN and EET (referred to using acronyms denoting the haploid genomes of their parent species), and their parental species. Results: Cobitis triploid hybrids exhibited intermediate karyotypes with ribosome synthesis sites on chromosomes inherited from both parents, showing no evidence of nucleolar dominance. The rDNA pattern derived from the C. elongatoides genome was more stable in the hybrids’ karyotypes. Two and one submetacentric chromosomes with co-localized rDNAs were effective markers to ascertain C. elongatoides diploid (EE) and haploid (E) genomes within the genome of triploid hybrids, respectively. Fewer 5S rDNA loci were observed in diploid (TT) and haploid (T) chromosome sets from C. taenia in ETT and EET females. C. taenia and C. tanaitica exhibited similar rDNA distribution patterns. Conclusions: The karyotypes of triploid Cobitis hybrids reflect the genomic contributions of their parental species. Variability in rDNA distribution patterns suggests complex genomic interactions in Cobitis hybrids resulting from polyploidization and hybridization, potentially influencing their reproductive potential. Full article

(This article belongs to the Special Issue Fish Cytogenetics: Insights into Genome Diversity)

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The karyotypes of triploid hybrid females of Cobitis. (A) Karyotype of female 3n = 73 (ETT) divided into chromosomes representing the parental species, Cobitis elongatoides (n = 25) and Cobitis taenia (2n = 48). (B) Karyotype of female 3n = 75 (EEN) divided into chromosomes representing the parental species, C. elongatoides (2n = 50) and Cobitis tanaitica (2n = 25). (C) Karyotype of female 3n = 74 (ETN) divided into chromosomes representing the parental species, C. elongatoides (n = 25), C. taenia (n = 24) and C. tanaitica (n = 25). (D) Karyotype of Cobitis female 3n = 74 * (EET) divided into chromosomes representing the parental species, C. elongatoides (2n = 50) and C. taenia (n = 24). Full article ">Figure 2
Double fluorescence in situ hybridization (FISH) with 28S and 5S rDNA probes: (A–C) Metaphase plate of hybrid Cobitis female (ETT, 3n = 73). (D) rDNA-bearing chromosomes, with numbering of chromosome pairs in the karyotype of Cobitis elongatoides (on the left) and Cobitis taenia (on the right) as parental species. (E–G) Metaphase plates of hybrid Cobitis female (EEN, 3n = 75). (H) rDNA-bearing chromosomes, with numbering of chromosome pairs in the karyotype of C. elongatoides (on the left) and unnumbered chromosomes of Cobitis tanaitica (on the right) as parental species. (C,G) Chromosomes with syntenic location of both 28S and 5S ribosomal genes; co-localizations of rDNA sites are indicated by arrows. Full article ">Figure 3
Double fluorescence in situ hybridization (FISH) with 28S and 5S rDNA probes: (A–C) Metaphase plate of hybrid Cobitis female (ETN, 3n = 74). (D) rDNA-bearing chromosomes, with numbering of chromosome pairs in the karyotype of Cobitis elongatoides (on the left) and unnumbered chromosomes of both Cobitis taenia and Cobitis tanaitica (on the right) as parental species. (E–G) Metaphase plates of Cobitis hybrid female (EET, 3n = 74 *). (H) rDNA-bearing chromosomes, with numbering of chromosome pairs in the karyotype of C. elongatoides (on the left) and C. taenia (on the right) as parental species. (C,G) Chromosomes with syntenic location of both 28S and 5S ribosomal genes; co-localizations of rDNA sites are indicated by arrows. Full article ">Figure 4
Schematic representation of the 5S (green) and 28S (red) rDNA distribution patterns in the karyotypes of triploid hybrid females of Cobitis of four different genome compositions (ETT, ETN, EET and EEN) divided into bi-armed chromosomes characteristic in the Cobitis elongatoides karyotype, uni-armed chromosomes characteristic in the Cobitis taenia karyotype (with numbering of chromosome pairs in the karyotype of these both parental species according to Grabowska et al. [<a href="#B8-genes-16-00068" class="html-bibr">8</a>]) and unnumbered uni-armed chromosomes in the Cobitis tanaitica (N) karyotype. Full article ">

11 pages, 1353 KiB

Open AccessArticle

Concordant Patterns of Population Genetic Structure in Food-Deceptive Dactylorhiza Orchids

by Ada Wróblewska, Beata Ostrowiecka, Edyta Jermakowicz and Izabela Tałałaj

Genes 2025, 16(1), 67; https://doi.org/10.3390/genes16010067 - 8 Jan 2025

Viewed by 327

Abstract

Background: The patterns of inbreeding coefficients (F_IS) and fine spatial genetic structure (FSGS) were evaluated regarding the mating system and inbreeding depression of food-deceptive orchids, Dactylorhiza majalis, Dactylorhiza incarnata var. incarnata, and Dactylorhiza fuchsii, from NE Poland. [...] Read more.

Background: The patterns of inbreeding coefficients (F_IS) and fine spatial genetic structure (FSGS) were evaluated regarding the mating system and inbreeding depression of food-deceptive orchids, Dactylorhiza majalis, Dactylorhiza incarnata var. incarnata, and Dactylorhiza fuchsii, from NE Poland. Methods: We used 455 individuals, representing nine populations of three taxa and AFLPs, to estimate percent polymorphic loci and Nei’s gene diversity, which are calculated using the Bayesian method; F_IS; F_ST; FSGS with the pairwise kinship coefficient (Fij); and AMOVA in populations. Results: We detected a relatively high proportion of polymorphic fragments (40.4–68.4%) and Nei’s gene diversity indices (0.140–0.234). The overall F_IS was relatively low to moderate (0.071–0.312). The average Fij for the populations of three Dactylorhiza showed significantly positive values, which were observed between plants at distances of 1–10 m (20 m). F_ST was significant in each Dactylorhiza taxon, ranging from the lowest values in D. fuchsii and D. majalis (0.080–0.086, p < 0.05) to a higher value (0.163, p < 0.05) in D. incarnata var. incarnata. Molecular variance was the highest within populations (76.5–86.6%; p < 0.001). Conclusions: We observed concordant genetic diversity patterns in three food-deceptive, allogamous, pollinator-dependent, and self-compatible Dactylorhiza. F_IS is often substantially higher than Fij with respect to the first class of FSGSs, suggesting that selfing (meaning of geitonogamy) is at least responsible for homozygosity. A strong FSGS may have evolutionary consequences in Dactylorhiza, and combined with low inbreeding depression, it may impact the establishment of inbred lines of D. majalis and D. incarnata var. incarnata. Full article

(This article belongs to the Special Issue Feature Papers in Population and Evolutionary Genetics and Genomics 2024)

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27 pages, 4250 KiB

Open AccessArticle

The RAD6-like Ubiquitin Conjugase Gene OsUBC7 Has a Positive Role in the Early Cold Stress Tolerance Response of Rice

by Huy Phan and Michael Schläppi

Genes 2025, 16(1), 66; https://doi.org/10.3390/genes16010066 - 8 Jan 2025

Viewed by 339

Abstract

Background/Objectives: Cold stress poses a significant threat to Asian rice cultivation, disrupting important physiological processes crucial for seedling establishment and overall plant growth. It is, thus, crucial to elucidate genetic pathways involved in cold stress tolerance response mechanisms. Methods: We mapped OsUBC7, [...] Read more.

Background/Objectives: Cold stress poses a significant threat to Asian rice cultivation, disrupting important physiological processes crucial for seedling establishment and overall plant growth. It is, thus, crucial to elucidate genetic pathways involved in cold stress tolerance response mechanisms. Methods: We mapped OsUBC7, a Radiation-sensitive 6 (RAD6)-type homolog of rice, to a low-temperature seedling survivability (LTSS) QTL and used genomics, molecular genetics, and physiological assays to assess its role in plant resilience against low-temperature stress. Results: OsUBC7 is cold responsive and has higher expression levels in cold-tolerant japonica than cold-sensitive indica. Overexpression of OsUBC7 enhances LTSS of indica and freezing tolerance of Arabidopsis, increases levels of soluble sugars and chlorophyll A, boosts leaf development after cold exposure, and increases leaf cell numbers and plants size, but it does not affect membrane stability after cold stress exposure. Additionally, OsUBC7 has a positive role for germinability in the presence of salt and for flowering and yield-related traits. The OsUBC7 protein physically interacts with the developmental stage-specific and histone-modifying E3 ligases OsRFPH2-12 and OsHUB1/2, respectively, and potential target genes such as cell cycle dependent kinases were identified. Conclusions: OsUBC7 might contribute to cold resilience by regulating sugar metabolism to provide energy for promoting cellular homeostasis restoration after cold stress exposure via new cell growth, particularly in leaf cells crucial for photosynthesis and metabolic activity, possibly by interacting with cell cycle regulating proteins. Overall, the present study suggests that OsUBC7 may be involved in plant development, reproduction, and stress adaptation, and contributes to a deeper understanding of rice plant cold stress tolerance response mechanisms. OsUBC7 may be a promising candidate for improving crop productivity and resilience to stressful environments. Full article

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

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Single-nucleotide polymorphisms (SNPs) and haplotypes of OsUBC7. (A) 11 SNPs of OsUBC7 in the 354 accessions of the Rice Diversity Panel 1 correlate with low-temperature seedling survivability (LTSS) scores. Two-week-old seedlings of the 354 accessions were exposed to constant 10 °C for 7 days and allowed to recover at warm temperatures for 7 days (28/25 °C day/night), after which LTSS was determined. (B) Haplotype analysis of OsUBC7 using RiceVarMap [<a href="#B33-genes-16-00066" class="html-bibr">33</a>] data from a population of 4402 rice accessions. (C) Haplotype–LTSS correlation analysis based on three major haplotypes of mentioned accessions. p values for Two-Way ANOVA: (**) p < 0.01; (***) p < 0.001. Full article ">Figure 2
Gene expression of OsUBC7 and its paralogs in different rice accessions and different temperature conditions. (A–D) Time series of OsUBC7 mRNA abundance in 2-week-old seedlings under warm control and low-temperature exposure (10 °C for aus Kasalath, 4 °C for temperate japonica Krasnodarskij 3352). Four housekeeping genes, 18S ribosomal RNA, OsACT1, OsUBC32, and OsUBQ5, corresponding to panels A, B, C, and D, respectively, were used for normalization. (E,F) mRNA abundance of OsUBC7, OsUBC8, and OsUBC9 in leaf tissues of Kasalath and Krasnodarskij 3352 at the 2-week-old seedling stage and in flag leaves at the flowering stage. 18S ribosomal RNA (panel E) and OsACT1 (panel F) were used for normalization. (*) p ≤ 0.05, (**) p ≤ 0.01 (Welch’s t-test). Full article ">Figure 3
Phenotype of OsUBC7 overexpression (OE) rice lines and aus Kasalath wild-type (WT) plants. (Top left) Two-week-old seedlings of OsUBC7 OE lines and WT plants before cold treatment. (Bottom left) Two-week-old seedlings of OsUBC7 OE lines and WT plants after cold treatment and 4 days of recovery. (Top right) OsUBC7 OE-1 line and segregated WT-1 plants after 5 days of recovery from cold treatment. (Bottom right) OsUBC7 OE line OE-2 and segregated WT-2 plants after 5 days of recovery from cold treatment. Full article ">Figure 4
Cold stress-related physiological phenotypes of OsUBC7 overexpression (OE) transgenic lines and wild-type (WT) plants. (A) Low-temperature seedling survivability (LTSS) of OsUBC7 OE and WT rice plants. (B) LTSS of OsUBC7 OE Arabidopsis, Col-0 WT, and AtUBC2 (OsUBC7 homolog) knockout (KO) plants exposed to −3 °C and recovering over a 7-day period. (C) Growth rate before cold treatment of OsUBC7 OE and WT rice plants. (D) Growth rate after cold treatment of OsUBC7 OE and WT rice plants. (E) Days to third leaf emergence after cold stress. (F) Plant height of 14-day-old OsUBC7 OE and WT seedlings. (α, ß, γ, δ) significant difference in OE or AtUBC2 KO lines compared to their respective WT plants. p values for Two-Way ANOVA: (*) p < 0.05; (**) p < 0.01; (ns) no significance. Full article ">Figure 5
Effect of OsUBC7 overexpression (OE) in transgenic plants on cold stress-related metabolites. (A) Soluble sugar content in OsUBC7 OE rice lines and wild-type (WT) plants. (B) Soluble sugar content in OsUBC7 OE Arabidopsis lines, WT Col-0, and AtUBC2 knockout (KO) plants. (C) Mean % electrolyte leakage levels in different tissues of OsUBC7 OE rice lines and WT plants after cold exposure. (D) Malondialdehyde content in different tissues of OsUBC7 OE rice lines and WT plants after cold exposure. (E) Electrolyte leakage in OsUBC7 OE Arabidopsis lines and WT plants. (α, ß, γ, δ) significance detected in OsUBC7 OE transgenic or AtUBC2 KO lines compared to their corresponding WT lines. p < 0.05, Two-Way ANOVA. DAS, day(s) after stress; ns, not significant. Full article ">Figure 6
Effect of OsUBC7 overexpression (OE) on leaf cell dimensions and cell density. (A) Leaf tissue of OsUBC7 OE and segregated wild-type (WT) lines stained with propidium iodide. (B) Cell density of OsUBC7 OE and WT lines. (C) Cell lengths of OsUBC7 OE and WT lines. (D) Cell widths of OsUBC7 OE and WT lines. p-values for Two-Way ANOVA: (*) < 0.05; (**) < 0.01; (ns) no significance. Full article ">Figure 7
Architecture of the first flag leaf and chlorophyll content of OsUBC7 overexpressing (OE) and wild-type (WT) lines. (A) Half-length of the first flag leaf, from the leaf tip to the middle. (B) Width of the first flag leaf. (C) Dry weight of the first flag leaf. (D) Total chlorophyll A content extracted from the entire flag leaf. (E) Total chlorophyll B content extracted from the entire flag leaf. p-values for Two-Way ANOVA: (*) < 0.05; (**) < 0.01; (ns) no significance. Full article ">Figure 8
Yeast two-hybrid assay showing interaction between Gal4-DNA binding domain (BD)-OsUBC7 and Gal4-Activation domain (AD)-E3 ligase fusions of OsRFPH2-12 (Left) and OsHUB1/HUB2 (Right; RING finger domain only). Positive interactions are shown by robust colony growth on quadruple dropout (QDO) selective plates, as seen for OsUBC7::RFPH2-12 and OsUBC7::OsHUB1/HUB2 in rows labeled “P”. A positive control showing interaction between a Gal4-BD-murine P53 fusion (bait) and a Gal4-AD-SV40 large T-antigen fusion (prey) is seen in rows labeled “+”. Negative controls of yeast transformed with the OsUBC7 bait and an empty prey vector (and vice versa) have no growth on QDO selective plates, as shown in rows labeled “–”. Bait and prey plasmids have robust growth on double dropout (DDO) selective plates. Full article ">

14 pages, 3211 KiB

Open AccessArticle

Transcriptome Analysis of Muscle Growth-Related circRNA in the Pacific Abalone Haliotis discus hanna

by Jianfang Huang, Jian He, Zhenghan She, Mingcan Zhou, Dongchang Li, Jianming Chen and Caihuan Ke

Genes 2025, 16(1), 65; https://doi.org/10.3390/genes16010065 - 8 Jan 2025

Viewed by 319

Abstract

(1) Background: Animal growth is a complex process, involving the coordination of a wide variety of genes, non-coding RNAs, and pathways. Circular RNAs (circRNAs) belong to a novel class of functional non-coding RNAs (ncRNAs). They have a distinctive ring structure and are involved [...] Read more.

(1) Background: Animal growth is a complex process, involving the coordination of a wide variety of genes, non-coding RNAs, and pathways. Circular RNAs (circRNAs) belong to a novel class of functional non-coding RNAs (ncRNAs). They have a distinctive ring structure and are involved in various biological processes, including the proliferation, differentiation, and apoptosis of muscle cells. The Pacific abalone Haliotis discus hannai is an economically valuable mollusk species cultivated in China. However, the modulation of muscle growth by circRNAs in this species is poorly understood. (2) Methods: In this study, we analyzed the muscle transcriptomes of 6 H. discus hannai specimens: three small (S_HD) and three large (L_HD) groups via RNA-seq and bioinformatics technology. (3) Results: The results indicated the presence of 11,744 circRNAs in abalone adductor muscle. Furthermore, the L_HD group had 250 significantly differentially expressed circRNAs (106 upregulated and 144 downregulated) relative to the S_HD group. Moreover, the bioinformatics assessment revealed that circRNAs were related to lipid transporter activity, lipid biosynthetic process, fat digestion and absorption, the single-organism metabolic process, the thyroid hormone signaling pathway, and the hippo signaling pathway, which regulates growth. Seventeen key candidate circRNAs were identified, and a core functional circRNA-miRNA-mRNA network associated with abalone muscle growth was described. Gene expression was verified using qRT-PCR, confirming the accuracy of the RNA-seq data. (4) Conclusion: Overall, this investigation furnishes novel evidence for the potential muscle growth modulatory mechanisms in Pacific abalone. These high-quality circRNA data of abalone muscle provide a reference for functional studies on the abalone genome. Full article

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

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The workflow of identifying circRNA. Full article ">Figure 2
Identification and features of circRNAs. (a) The Venn diagram of circRNAs. (b) The length distribution of most circRNAs. (c) The genomic location of circRNAs. Full article ">Figure 3
Heatmaps and volcano plots of differentially expressed circRNAs (DE-circRNAs). (a) CircRNA expression in large (L_HD) vs. small (S_HD) abalone group. Red and green dots indicate up- and downregulated circRNAs, respectively. (b) Hierarchical clustering of DE-circRNAs. Red rectangles represent upregulated circRNAs; blue rectangles represent downregulated circRNAs. Full article ">Figure 4
Competitive endogenous RNA (ceRNA) network of circRNA-miRNA-mRNA interactions in the S_HD and L_HD groups. (a) Relationship between DE-circRNA and DE-mRNA. (b) The interaction between miRNA and circRNAs. The miRNAs are shown in blue. The mRNAs are represented in green. The circRNAs are represented in red. Full article ">Figure 5
Relative expression of DE-circRNAs quantified with qRT-PCR. (a–f) The relative expression levels of novel_circ_0007575, novel_circ_0008967, novel_circ_0008580, novel_circ_0003383, novel_circ_0003381, and novel_circ_0003380, respectively. Data are shown as mean ± SD (n = 3). *, p < 0.05; **, p < 0.01. Full article ">

33 pages, 1758 KiB

Open AccessArticle

Quantitative Trait Loci for Phenology, Yield, and Phosphorus Use Efficiency in Cowpea

by Saba B. Mohammed, Patrick Obia Ongom, Nouhoun Belko, Muhammad L. Umar, María Muñoz-Amatriaín, Bao-Lam Huynh, Abou Togola, Muhammad F. Ishiyaku and Ousmane Boukar

Genes 2025, 16(1), 64; https://doi.org/10.3390/genes16010064 - 8 Jan 2025

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Abstract

Background/Objectives: Cowpea is an important legume crop in sub-Saharan Africa (SSA) and beyond. However, access to phosphorus (P), a critical element for plant growth and development, is a significant constraint in SSA. Thus, it is essential to have high P-use efficiency varieties to [...] Read more.

Background/Objectives: Cowpea is an important legume crop in sub-Saharan Africa (SSA) and beyond. However, access to phosphorus (P), a critical element for plant growth and development, is a significant constraint in SSA. Thus, it is essential to have high P-use efficiency varieties to achieve increased yields in environments where little-to- no phosphate fertilizers are applied. Methods: In this study, crop phenology, yield, and grain P efficiency traits were assessed in two recombinant inbred line (RIL) populations across ten environments under high- and low-P soil conditions to identify traits’ response to different soil P levels and associated quantitative trait loci (QTLs). Single-environment (SEA) and multi-environment (MEA) QTL analyses were conducted for days to flowering (DTF), days to maturity (DTM), biomass yield (BYLD), grain yield (GYLD), grain P-use efficiency (gPUE) and grain P-uptake efficiency (gPUpE). Results: Phenotypic data indicated significant variation among the RILs, and inadequate soil P had a negative impact on flowering, maturity, and yield traits. A total of 40 QTLs were identified by SEA, with most explaining greater than 10% of the phenotypic variance, indicating that many major-effect QTLs contributed to the genetic component of these traits. Similarly, MEA identified 23 QTLs associated with DTF, DTM, GYLD, and gPUpE under high- and low-P environments. Thirty percent (12/40) of the QTLs identified by SEA were also found by MEA, and some of those were identified in more than one P environment, highlighting their potential in breeding programs targeting PUE. QTLs on chromosomes Vu03 and Vu08 exhibited consistent effects under both high- and low-P conditions. In addition, candidate genes underlying the QTL regions were identified. Conclusions: This study lays the foundation for molecular breeding for PUE and contributes to understanding the genetic basis of cowpea response in different soil P conditions. Some of the identified genomic loci, many being novel QTLs, could be deployed in marker-aided selection and fine mapping of candidate genes. Full article

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

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Frequency distributions of phenotypic data under high-and low-P environments in the TVu-14676 x IT84S-2246 population. High P is depicted in red colour, low P in sky blue, and blue-grey is the overlap between environments. The horizontal and vertical axes represent the trait value and number of genotypes, respectively. DTF.E1–E4 = days to flowering for Environment 1 to 4, DTM.E1–E4 = days to maturity for Environment 1 to 4, BYLD.E1–E5 = biomass yield for Environment 1 to 5, GYLD.E1–E5 = grain yield (kg/ha) for Environment 1–5, gPUE.E1–E2 = P-use efficiency for Environment 1 and 2, and gPUpE.E1–E2 = P-uptake efficiency of grain for Environment 1 and 2. E1 = 2017.Zaria, E2 = 2018.Zaria, E3 = 2018.Minjibir, E4 = 2018.Mokwa and E5 = 2018.Kadawa. Full article ">Figure 2
Frequency distributions of phenotypic data under high-P and low-P environments in the Yacine × 58-77 population. High P is illustrated in red colour, low P in sky blue, and blue-grey is the overlap between environments. The horizontal and vertical axes represent the trait value and number of genotypes, respectively. DTF.E1–4 = days to flowering for Environment 1 to 4, DTM.E1–4 = days to maturity for Environment 1 to 4, BYLD.E1–5 = biomass yield for Environment 1 to 5, GYLD.E1–5 = grain yield (kg/ha) for Environment 1–5, gPUE.E1–2 = P-use efficiency for Environment 1 and 2 and gPUpE.E1–2 = P-uptake efficiency for Environment 1 and 2. E1 = 2017.Zaria, E2 = 2018.Zaria, E3 = 2018.Minjibir, E4 = 2018.Mokwa and E5 = 2018.Kadawa. Full article ">Figure 3
Chromosome-wide distribution of the identified QTLs in comparison with previous studies. QTL regions are shown by chromosome. cM positions are on the left, with some positions skipped. SEA QTLs from the present study are represented in green, MEA QTLs are represented in red, and QTLs from other studies are shown in blue. Full article ">

16 pages, 3031 KiB

Open AccessArticle

Genome-Wide Insights into Internalizing Symptoms in Admixed Latin American Children

by Gabriela de Sales Guerreiro Britto, Alberto O. Moreira, Edson Henrique Bispo Amaral, Daniel Evangelista Santos, Raquel B. São Pedro, Thaís M. M. Barreto, Caroline Alves Feitosa, Darci Neves dos Santos, Eduardo Tarazona-Santos, Maurício Lima Barreto, Camila Alexandrina Viana de Figueiredo, Ryan dos Santos Costa, Ana Lúcia Brunialti Godard and Pablo Rafael Silveira Oliveira

Genes 2025, 16(1), 63; https://doi.org/10.3390/genes16010063 - 8 Jan 2025

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Abstract

Background/Objectives: Internalizing disorders, including depression and anxiety, are major contributors to the global burden of disease. While the genetic architecture of these disorders in adults has been extensively studied, their early-life genetic mechanisms remain underexplored, especially in non-European populations. This study investigated the [...] Read more.

Background/Objectives: Internalizing disorders, including depression and anxiety, are major contributors to the global burden of disease. While the genetic architecture of these disorders in adults has been extensively studied, their early-life genetic mechanisms remain underexplored, especially in non-European populations. This study investigated the genetic mechanisms underlying internalizing symptoms in a cohort of Latin American children. Methods: This study included 1244 Brazilian children whose legal guardians completed the Child Behavior Checklist (CBCL) questionnaire. Genotyping was performed using the Illumina HumanOmni 2.5-8v1 BeadChip. Results: The genome-wide association analysis revealed a significant association of rs7196970 (p = 4.5 × 10⁻⁸, OR = 0.61), in the ABCC1 gene, with internalizing symptoms. Functional annotation highlighted variants in epigenetically active regulatory regions, with multiple variants linked to differential expression of ABCC1 across several human tissues. Pathway enrichment analysis identified 42 significant pathways, with notable involvement in neurobiological processes such as glutamatergic, GABAergic, and dopaminergic synapses. Conclusions: This study identifies ABCC1 variants as novel genetic factors potentially associated with early-life internalizing symptoms. These results may contribute to future research on targeted interventions for childhood internalizing conditions. Full article

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

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Ancestry analyses of children from the SCAALA cohort. (A) Principal Component Analysis (PCA) comparing children from the SCAALA cohort with reference populations from the 1000 Genomes Project. (B) Bar plots showing the individual ancestries of the participants (N-INT, n = 794 individuals; INT, n = 450), as determined by the ADMIXTURE method. Abbreviations: N-INT, CBCL T score < 64. INT, CBCL T score ≥ 64; Europeans (EUR), Native Americans (NAT), South Asians (SAS), East Asians (EAS), and Africans (AFR); IQR, interquartile range (first–third quartiles); p, the p-value for the Mann–Whitney test; ns, not significant. Full article ">Figure 2
Genome-wide association analysis of internalizing symptoms in children from the SCAALA cohort. (A) Quantile–Quantile (QQ) plot showing observed and expected p-values. (B) Manhattan plot of association statistics obtained from multivariate logistic regression (additive model), with sex and seven principal components included as covariates. The red line represents the genomic significance threshold (p < 5 × 10−8), while the blue line indicates the threshold for suggestive associations (5 × 10−8 < p < 10−5). (C) Regional association plot at the ABCC1 locus. The plot shows linkage disequilibrium (LD, r2) between the lead variant rs7196970 (purple diamond) and other variants (circles) within the region 16:15503151–16239180 (RefSeq: GRCh38). The positions of coding genes within this region are shown at the bottom of the figure. Full article ">Figure 3
Functional annotation of variants in linkage disequilibrium with rs7196970 (ABCC1 locus). (A) Schematic diagram of the locus containing the ABCC1 gene. The green solid lines and rectangles represent introns and exons, respectively. This region was cross-referenced with DNA sequence annotations, including short variants from the gnomAD consortium, previous associations with human traits (as recorded in the GWAS catalog platform), regulatory elements, and expression Quantitative Trait Locus (eQTL) data from the GTEx multi-tissue meta-analysis (p-value). (B) Magnified view of a region within the ABCC1 gene, highlighting SNVs in moderate to high linkage disequilibrium (r2 > 0.6) with rs7196970. Image generated using the Ensembl Genome Browser (<a href="http://www.ensembl.org" target="_blank">http://www.ensembl.org</a>, accessed on 15 July 2024). Full article ">Figure 4
Pathway enrichment analysis for internalizing symptoms in children from the SCAALA cohort. Overrepresentation analysis was conducted on 2122 genes, which were matched with the canonical KEGG pathways. Volcano plot showing the pathways. The red line represents the significance threshold [p-value from the false discovery ratio (pFDR) < 0.05]. The size of the dots corresponds to the pathway’s gene set size. The ratio of enrichment is the number of observed genes divided by the number of expected genes from KEGG (according to the WebGestalt tool, <a href="http://www.webgestalt.org/" target="_blank">www.webgestalt.org/</a>, accessed on 20 October 2024). Full article ">

13 pages, 1504 KiB

Open AccessArticle

Expanding the Molecular Spectrum of MMP21 Missense Variants: Clinical Insights and Literature Review

by Domizia Pasquetti, Paola Tesolin, Federica Perino, Stefania Zampieri, Marco Bobbo, Thomas Caiffa, Beatrice Spedicati and Giorgia Girotto

Genes 2025, 16(1), 62; https://doi.org/10.3390/genes16010062 - 8 Jan 2025

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Abstract

Background/Objectives: The failure of physiological left-right (LR) patterning, a critical embryological process responsible for establishing the asymmetric positioning of internal organs, leads to a spectrum of congenital abnormalities characterized by laterality defects, collectively known as “heterotaxy”. MMP21 biallelic variants have recently been associated [...] Read more.

Background/Objectives: The failure of physiological left-right (LR) patterning, a critical embryological process responsible for establishing the asymmetric positioning of internal organs, leads to a spectrum of congenital abnormalities characterized by laterality defects, collectively known as “heterotaxy”. MMP21 biallelic variants have recently been associated with heterotaxy syndrome and congenital heart defects (CHD). However, the genotype–phenotype correlations and the underlying pathogenic mechanisms remain poorly understood. Methods: Patients harboring biallelic MMP21 missense variants who underwent diagnostic genetic testing for CHD or heterotaxy were recruited at the Institute for Maternal and Child Health—I.R.C.C.S. “Burlo Garofolo”. Additionally, a literature review on MMP21 missense variants was conducted, and clinical data from reported patients, along with molecular data from in silico and modeling tools, were collected. Results: A total of 18 MMP21 missense variants were reported in 26 patients, with the majority exhibiting CHD (94%) and variable extra-cardiac manifestations (64%). In our cohort, through Whole-Exome Sequencing (WES) analysis, the missense p.(Met301Ile) variant was identified in two unrelated patients, who both presented with heterotaxy syndrome. Conclusions: Our comprehensive analysis of MMP21 missense variants supports the pathogenic role of the p.(Met301Ile) variant and provides significant insights into the disease pathogenesis. Specifically, missense variants are distributed throughout the gene without clustering in specific regions, and phenotype comparisons between patients carrying missense variants in compound heterozygosity or homozygosity do not reveal significant differences. These findings may suggest a potential loss-of-function mechanism for MMP21 missense variants, especially those located in the catalytic domain, and highlight their critical role in the pathogenesis of heterotaxy syndrome. Full article

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

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(A) Family pedigree of Patient 1, showing grade of parents’ consanguinity, (B) ECG of Patient 1 displaying multifocal atrial beats with junctional rhythm, right axis deviation and positive T wave in right precordial leads; (C) Family pedigree of Patient 2, (D) ECG of Patient 2 showing suggestive findings of ventricular inversion, including absence of q waves in V5–V6 and presence of q waves in V1. Filled symbols with black arrows represent index patients; half-filled symbols represent healthy carrier individuals; numbers in pedigree represent the number of individuals with the same degree of biological relationship (e.g., Patient 2 has two paternal uncles). Full article ">Figure 2
(A) Schematic representation of the MMP21 gene and the MMP21 protein. Exons are depicted in blue and introns in light blue. Protein domains are represented in different colors and aminoacidic positions at the beginning and end of each domain are reported. Missense variants identified in the literature are reported. (B) Structural model of MMP21 as predicted by AlphaFold (XZ plane). Protein backbone is represented in gray, while domains are reported in the same color as <a href="#genes-16-00062-f002" class="html-fig">Figure 2</a>A (i.e., ZnMc domain in green and HX domains in pink). Aminoacidic positions in which missense variants occur are colored in red in the 3D structure. (C) Backbone of the ZnMc domain of MMP21 is reported in green. Positions in which missense variants occur are colored in red and amino acid structures are highlighted (carbon atoms are depicted in grey, oxygen in red, nitrogen in blue, and sulfur in yellow). (D) Protein alignment showing conservation of methionine 301 across species, highlighted by the red frame; amino acids with similar physicochemical properties are represented in the same colors, as defined by the Clustal Omega software. (ZnMc: Zinc-dependent metalloprotease; HX: Hemopexin-like repeats). In figures, the variant identified in our patients, c.903G>A, p.(Met301Ile), is reported in red. Full article ">

18 pages, 14549 KiB

Open AccessArticle

Evolutionary and Structural Insights into the RNA Polymerase I A34 Protein Family: A Focus on Intrinsic Disorder and Phase Separation

by Bruce A. Knutson and Lawrence I. Rothblum

Genes 2025, 16(1), 61; https://doi.org/10.3390/genes16010061 - 7 Jan 2025

Viewed by 382

Abstract

Background: Eukaryotic RNA polymerase I consists of 12 or 11 core subunits and three dissociable subunits, Rrn3, A34, and A49. The A34 and A49 subunits exist as a heterodimer. In silico analysis of the A34 family of transcription factors demonstrates a commonly shared [...] Read more.

Background: Eukaryotic RNA polymerase I consists of 12 or 11 core subunits and three dissociable subunits, Rrn3, A34, and A49. The A34 and A49 subunits exist as a heterodimer. In silico analysis of the A34 family of transcription factors demonstrates a commonly shared domain structure despite a lack of sequence conservation, as well as N–terminal and C-terminal disordered regions. The common structure of A34 has an N–terminal disordered region followed by a dimerization domain that, in conjunction with A49, contributes to a fold that resembles the TFIIF core. This in turn is followed by a short region that cryo-EM demonstrates resembles an arm and intimately interacts with the PolR1A, PolR1B, and PolR1C subunits of Pol I. Analyses: This Pol I–binding domain is then followed by a region that is not resolved in cryo-EM and is predicted to be intrinsically disordered. Interestingly, the size/length of this disordered structure increases from yeast to humans, and is composed of repeats with unique sequence and biochemical features that also increase in number. Further analyses of the A34 CTD (carboxy–terminal domain) indicate that it has a high probability of undergoing liquid–liquid phase separation. Conclusions: We suggest that this intrinsically disordered domain found in the A34 family of Pol I transcription factors serves a function similar to the CTD of the PolR2A subunit in coordinating transcription initiation and elongation and RNA processing. Lastly, we propose that dynamic acetylation of PAF49 may regulate interactions of the intrinsically disordered CTD and thereby specify liquid–liquid phase separations. Overall, we propose a new paradigm for a repeat-containing CTD in Pol I transcription. Full article

(This article belongs to the Section Bioinformatics)

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13 pages, 1118 KiB

Open AccessArticle

Novel OTOG Variants and Clinical Features of Hearing Loss in a Large Japanese Cohort

by Yasuhiro Arai, Shin-ya Nishio, Shinichi Goto, Yumiko Kobayashi, Yohei Honkura, Akira Ganaha, Kotaro Ishikawa, Shin-ichiro Oka, Hiroshi Futagawa, Mayuri Okami, Fumio Takada, Kyoko Nagai, Tomoko Esaki, Takayuki Okano, Yumi Ohta, Shin Masuda, Kentaro Egusa, Masato Teraoka, Kazuma Sugahara and Shin-ichi Usami

Genes 2025, 16(1), 60; https://doi.org/10.3390/genes16010060 - 7 Jan 2025

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Abstract

Background/Objectives: The OTOG gene is responsible for autosomal recessive non-syndromic sensorineural hearing loss and is assigned as DFNB18B. To date, 44 causative OTOG variants have been reported to cause non-syndromic hearing loss. However, the detailed clinical features for OTOG-associated hearing loss remain [...] Read more.

Background/Objectives: The OTOG gene is responsible for autosomal recessive non-syndromic sensorineural hearing loss and is assigned as DFNB18B. To date, 44 causative OTOG variants have been reported to cause non-syndromic hearing loss. However, the detailed clinical features for OTOG-associated hearing loss remain unclear. Methods: In this study, we analyzed 7065 patients with non-syndromic hearing loss (mean age 26.4 ± 22.9 years, 2988 male, 3855 female, and 222 without gender information) using massively parallel DNA sequencing for 158 target deafness genes. We identified the patients with biallelic OTOG variants and summarized the clinical characteristics. Results: Among the 7065 patients, we identified 14 possibly disease-causing OTOG variants in 26 probands, with 13 of the 14 variants regarded as novel. Patients with OTOG-associated hearing loss mostly showed congenital or childhood-onset hearing loss. They were considered to show non-progressive, mild-to-moderate hearing loss. There were no symptoms that accompanied the hearing loss in OTOG-associated hearing loss patients. Conclusions: We confirmed non-progressive, mild-to-moderate hearing loss as the clinical characteristics of OTOG-associated hearing loss. These findings will contribute to a better understanding of the clinical features of OTOG-associated HL and will be useful in clinical practice. Full article

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

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14 pages, 280 KiB

Open AccessReview

Recent Advances in Stroke Genetics—Unraveling the Complexity of Cerebral Infarction: A Brief Review

by Takeshi Yoshimoto, Hiroshi Yamagami and Yuji Matsumaru

Genes 2025, 16(1), 59; https://doi.org/10.3390/genes16010059 - 6 Jan 2025

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Abstract

Background/Objectives: Recent advances in stroke genetics have substantially enhanced our understanding of the complex genetic architecture underlying cerebral infarction and other stroke subtypes. As knowledge in this field expands, healthcare providers must remain informed about these latest developments. This review aims to provide [...] Read more.

Background/Objectives: Recent advances in stroke genetics have substantially enhanced our understanding of the complex genetic architecture underlying cerebral infarction and other stroke subtypes. As knowledge in this field expands, healthcare providers must remain informed about these latest developments. This review aims to provide a comprehensive overview of recent advances in stroke genetics, with a focus on cerebral infarction, and discuss their potential impact on patient care and future research directions. Methods: We reviewed recent literature about advances in stroke genetics, focusing on cerebral infarction, and discussed their potential impact on patient care and future research directions. Key developments include the identification of monogenic stroke syndromes, such as cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy, and cerebral autosomal recessive arteriopathy with subcortical infarcts and leukoencephalopathy caused by mutations in the NOTCH3 and HTRA1 genes, respectively. In addition, the role of RNF213 in moyamoya disease and other cerebrovascular disorders, particularly in East Asian populations, has been elucidated. The development of polygenic risk scores for assessing genetic predisposition to stroke has demonstrated the potential to improve risk prediction beyond traditional factors. Genetic studies have also elucidated the distinct genetic architecture of stroke subtypes, including large artery atherosclerosis, small vessel disease, and cardioembolic stroke. Furthermore, the investigation of epigenetic modifications influencing stroke risk and its outcomes has revealed new research avenues, while advancements in pharmacogenomics highlight the potential for personalized stroke treatment based on individual genetic profiles. Conclusions: These genetic discoveries have important clinical implications, including improved risk stratification, targeted prevention strategies, and the development of novel therapeutic approaches. Full article

(This article belongs to the Special Issue Stroke Genomics and Exit Strategies)

13 pages, 5750 KiB

Open AccessArticle

Suppression of Nodule Formation by RNAi Knock-Down of Bax inhibitor-1a in Lotus japonicus

by Fuxiao Jin, Danxia Ke, Lu Lu, Qianqian Hu, Chanjuan Zhang, Chao Li, Wanwan Liang, Songli Yuan and Haifeng Chen

Genes 2025, 16(1), 58; https://doi.org/10.3390/genes16010058 - 6 Jan 2025

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Abstract

Background/Objectives: The balanced regulation of innate immunity plays essential roles in rhizobial infection and the establishment and maintenance of symbiosis. The evolutionarily conserved cell death suppressor Bax inhibitor-1 plays dual roles in nodule symbiosis, providing a valuable clue in balancing immunity and symbiosis, [...] Read more.

Background/Objectives: The balanced regulation of innate immunity plays essential roles in rhizobial infection and the establishment and maintenance of symbiosis. The evolutionarily conserved cell death suppressor Bax inhibitor-1 plays dual roles in nodule symbiosis, providing a valuable clue in balancing immunity and symbiosis, while it remains largely unexplored in the legume Lotus japonicus. Methods/Results: In the present report, the BI-1 gene family of L. japonicus was identified and characterized. We identified 6 BI-1 genes that translate into peptides containing 240–255 amino acids with different structural characteristics and isoelectric points. We performed phylogenetic analyses and detected evolutionary conservation and divergence among BI-1 proteins from L. japonicus, Glycine max, Medicago truncatula, Arabidopsis thaliana, and Oryza sativa. Expression profiles among different roots indicated that the inoculation of MAFF303099 significantly increased the expression of most of the L. japonicus BI-1 family genes. We down-regulated the transcripts of LjBI-1a by RNA interference and observed that LjBI-1a promotes nodulation and nodule formation. Conclusions: These discoveries shed light on the functions of BI-1 genes in L. japonicus, and simultaneously emphasize the potential application of LjBI-1a in enhancing the symbiotic nitrogen fixation ability of legumes. Full article

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

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Phylogenetic analysis of the BI-1 gene family in L. japonicus, Glycine max, Oryza sativa, Arabidopsis thaliana, and Medicago truncatula. The neighbor-joining phylogenetic tree was constructed using MEGA version 11.0 with a JTT + G model and 1000 bootstrap replicates. The tree divided these BI-1 proteins into three major groups (Group A to Group C) with different colors. The green, pink, and cyan colors represent the A–C groups, respectively. The different shapes in blue indicate different species. The red font represents subgroup C1: 9 legume plant BI-1 genes. Full article ">Figure 2
Alignments of the conserved motifs of the 9 BI-1 genes in subgroup C1. The red, green, and blue boxes are the main retention patterns. The red line represents the Bax inhibitor (BI)-1 protein family motif; the green dashed line represents the Integral membrane protein YbhL motif; and within the blue box is the Conjugal transfer coupling protein TraG motif. Full article ">Figure 3
Expression profile of LjBI-1 genes in L. japonicus roots with and without Rhizobium MAFF303099 inoculation at three post-inoculation time points. (A) LjBI-1a (B) LjBI-1b (C) LjBI-1c (D) LjBI-1d (E) LjBI-1e (F) LjBI-1f. The control (un-inoculated) and inoculated roots at 6 h, 30 h, and 3 d post-inoculation were used to extract RNA, and the specific primers of the six LjBI-1 genes were utilized to perform qPCR. The expression levels of 6 LjBI-1 genes were detected using three biological replicate samples. qPCR was used to obtain the relative expression levels of each LjBI-1 gene, which were then normalized to the average expression level of the L. japonicus reference gene QACT. The expression levels in un-inoculated roots at the same time point were used as the controls for calculation. These results represent the mean ± SD of three independent biological repetitions. Asterisks represent significant differences, as determined by Student’s t-test (** p < 0.01; * 0.01 < p < 0.05; ns: p > 0.05). Full article ">Figure 4
Nodulation phenotype of LjBI-1a RNAi in L. japonicus. (A) Photographed images representing the hairy root systems expressing the empty vector (control), LjBI-1a RNAi-1, and LjBI-1a RNAi-2. Photographs were taken 30 days after inoculation with M. loti MAFF303099, and plants were grown without nitrogen fertilizer. Bars = 10 mm. Mean number of nodules (B), mean root length (C), and mean root fresh weight (D) per plant with standard deviation (SD) of L. japonicus expressing the empty vector (control), LjBI-1a RNAi-1, and LjBI-1a RNAi-2 at 30 days post-inoculation with M. loti. (E) qPCR analysis of transcript levels of LjBI-1a, NIN, Enod40, and Lb in the control, LjBI-1a RNAi-1, and LjBI-1a RNAi-2 hairy roots. These results represent the mean ± SD of three independent biological repetitions. Asterisks represent significant differences, as determined by Student’s t-test (** p < 0.01; * 0.01 < p < 0.05; ns: p > 0.05). Full article ">Figure 5
IT numbers at each infection stage in hairy roots ten days after Rhizobium inoculation. Empty vector served as control. Each root system was analyzed using twenty roots with lengths of 4–6 cm. Full article ">

13 pages, 1545 KiB

Open AccessArticle

The Heterozygous p.A684V Variant in the WFS1 Gene Is a Mutational Hotspot Causing a Severe Hearing Loss Phenotype

by Shintaro Otsuka, Chihiro Morimoto, Shin-ya Nishio, Shinya Morita, Daisuke Kikuchi, Masahiro Takahashi, Kozo Kumakawa, Yasuhiro Arai, Hajime Sano, Hidekane Yoshimura, Norio Yamamoto, Shunsuke Kondo, Mari Hasegawa, Tomo Nishi, Tadashi Kitahara and Shin-ichi Usami

Genes 2025, 16(1), 57; https://doi.org/10.3390/genes16010057 - 6 Jan 2025

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Abstract

Background/Objectives: A heterozygous mutation in the WFS1 gene is responsible for autosomal dominant non-syndromic hearing loss (DFNA6/14/38) and Wolfram-like syndrome, which is characterized by bilateral sensorineural hearing loss with optic atrophy and/or diabetes mellitus. However, detailed clinical features for the patients with the [...] Read more.

Background/Objectives: A heterozygous mutation in the WFS1 gene is responsible for autosomal dominant non-syndromic hearing loss (DFNA6/14/38) and Wolfram-like syndrome, which is characterized by bilateral sensorineural hearing loss with optic atrophy and/or diabetes mellitus. However, detailed clinical features for the patients with the heterozygous p.A684V variant remain unknown. Methods: We report the clinical details of 14 cases with a heterozygous p.A684V variant in the WFS1 gene identified from target resequencing analysis of 63 previously reported deafness genes by next-generation sequencing of 15,684 hearing loss patients (mean age 27.5 ± 23.1 years old, 6574 male, 8612 female and 498 for whom information was unavailable). Results: Among the 14 patients from 13 families with the p.A684V variant, nine were sporadic cases. In addition, we confirmed de novo occurrence of this variant in seven families. This result strongly supports the notion that this variant was located on a mutational hotspot. When comparing previously reported cases of autosomal dominant WFS1 gene-associated hearing loss, most of the patients in this study showed severe-to-profound bilateral sensorineural hearing loss (genotype–phenotype correlation). Two patients had optic atrophy, while the others did not have any other complications. Conclusions: The identified heterozygous p.A684V variant appears to be a hotspot mutation and likely to cause severe-to-profound hearing loss in early childhood. Cochlear implantation is considered favorable in cases of hearing impairment due to this variant. Full article

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

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15 pages, 4739 KiB

Open AccessArticle

Pan-Cancer Upregulation of the FOXM1 Transcription Factor

by Daniele Pozzobon, Arianna Bellezza and Federico M. Giorgi

Genes 2025, 16(1), 56; https://doi.org/10.3390/genes16010056 - 6 Jan 2025

Viewed by 380

Abstract

Background: The human FOXM1 transcription factor controls cell cycle progression and genome stability, and it has been correlated to the onset and progression of many tumor types. Methods: In our study, we collected all recent sequence and quantitative transcriptomics data about FOXM1, testing [...] Read more.

Background: The human FOXM1 transcription factor controls cell cycle progression and genome stability, and it has been correlated to the onset and progression of many tumor types. Methods: In our study, we collected all recent sequence and quantitative transcriptomics data about FOXM1, testing its presence across vertebrate evolution and its upregulation in cancer, both in bulk tissue contexts (by comparing the TCGA tumor dataset and the GTEx normal tissue dataset) and in single-cell contexts. Results: FOXM1 is significantly and consistently upregulated in all tested tumor types, as well as in tumor cells within a cancer microenvironment. Its upregulation reverberates in the upregulation of its target genes and can be used as a biomarker for poor cancer outcome in at least four tumor types. Conclusions: Despite its lack of cancer-related mutations and amplifications, the recurring upregulation of FOXM1 in all tumors puts a focusing lens on this gene as a candidate pan-cancer master regulator. Full article

(This article belongs to the Special Issue Genomic Diagnosis of Human Cancers)

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(A) Domain annotation of human FOXM1 isoforms. The Forkhead (FH) domain is shown as an octagon. Purple boxes indicate low complexity regions. Vertical bars are provided by the SMART tool for the most studied isoforms and indicate the positions of intron/exon junction. (B) Genomic alteration detection for FOXM1 across the TCGA Pan-Cancer Atlas (calculated by the cBioPortal OncoPrint algorithm). (C) Location of single-point somatic mutations in the FOXM1 gene according to the COSMIC cancer catalogue. Full article ">Figure 2
Phylogenetic tree of FOXM1 orthologs in vertebrates. The optimal tree, calculated using the Neighbor-Joining method, is shown. 100 bootstrapping replicates were generated, and the percentage of replicate trees in which the downstream taxa grouped together is shown. The branch lengths are proportional to the number of amino acid substitutions per site, after the Poisson correction method was applied. Tree drawn with MEGA11 and Inkscape. Full article ">Figure 3
Expression of FOXM1 across 15 human normal (GTEx) and cancer (TCGA) tissues. (A) Box plots indicting the difference between tumor (left) and normal (right) samples. (B) Beeswarm plots indicating gene-by-gene differential expression (expressed as DESeq2 negative binomial statistics) across 15 human cancer vs. normal comparisons. The X indicates the position of FOXM1 in the gene ranking, and the number below each plot indicates the ranking of FOXM1 in the upregulated transcriptome. Full article ">Figure 4
(A) Survival analysis of FOXM1 for the 15 TCGA cancer types selected in this study. Patients were stratified in two groups: with FOXM1 expression above the mean (“FOXM1 high”, in red) and below the mean (“FOXM1 low”, in blue). (B) Master regulator analysis of FOXM1 across the 15 TCGA cancer types selected in this study. Full article ">Figure 5
FOXM1 in single cancer cells. (A) Beeswarm plots indicating the levels of FOXM1 in normal cells (left) and tumor cells (right). (B) UMAP plots indicating the expression level of FOXM1 (as LogScale-normalized FPKMs) in normal and tumor cells. Full article ">

35 pages, 2960 KiB

Open AccessReview

Lipoprotein Lipase: Structure, Function, and Genetic Variation

by Shehan D. Perera, Jian Wang, Adam D. McIntyre and Robert A. Hegele

Genes 2025, 16(1), 55; https://doi.org/10.3390/genes16010055 - 5 Jan 2025

Viewed by 550

Abstract

Biallelic rare pathogenic loss-of-function (LOF) variants in lipoprotein lipase (LPL) cause familial chylomicronemia syndrome (FCS). Heterozygosity for these same variants is associated with a highly variable plasma triglyceride (TG) phenotype ranging from normal to severe hypertriglyceridemia (HTG), with longitudinal variation in [...] Read more.

Biallelic rare pathogenic loss-of-function (LOF) variants in lipoprotein lipase (LPL) cause familial chylomicronemia syndrome (FCS). Heterozygosity for these same variants is associated with a highly variable plasma triglyceride (TG) phenotype ranging from normal to severe hypertriglyceridemia (HTG), with longitudinal variation in phenotype severity seen often in a given carrier. Here, we provide an updated overview of genetic variation in LPL in the context of HTG, with a focus on disease-causing and/or disease-associated variants. We provide a curated list of 300 disease-causing variants discovered in LPL, as well as an exon-by-exon breakdown of the LPL gene and protein, highlighting the impact of variants and the various functional residues of domains of the LPL protein. We also provide a curated list of variants of unknown or uncertain significance, many of which may be upgraded to pathogenic/likely pathogenic classification should an additional case and/or segregation data be reported. Finally, we also review the association between benign/likely benign variants in LPL, many of which are common polymorphisms, and the TG phenotype. Full article

(This article belongs to the Special Issue Genetic and Genomic Research of Cardiovascular Diseases)

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16 pages, 700 KiB

Open AccessReview

Pharmacogenetics of the Treatment of Neglected Diseases

by Tiffany Borges Cabral, Amanda Carvalho de Oliveira, Gisely Cardoso de Melo and Fernanda Rodrigues-Soares

Genes 2025, 16(1), 54; https://doi.org/10.3390/genes16010054 - 5 Jan 2025

Viewed by 575

Abstract

Background/Objectives: Pharmacogenetics (PGx) aims to identify individuals more likely to suffer from adverse reactions or therapeutic failure in drug treatments. However, despite most of the evidence in this area being from European populations, some diseases have also been neglected, such as HIV infection, [...] Read more.

Background/Objectives: Pharmacogenetics (PGx) aims to identify individuals more likely to suffer from adverse reactions or therapeutic failure in drug treatments. However, despite most of the evidence in this area being from European populations, some diseases have also been neglected, such as HIV infection, malaria, and tuberculosis. With this review, we aim to emphasize which pharmacogenetic tests are ready to be implemented in treating neglected diseases that have some evidence and call attention to what is missing for these three diseases. Methods: A critical literature review on the PGx of HIV infection, malaria, and tuberculosis was performed. Results: There are three PGx guidelines for antiretroviral drugs used in HIV infection, one for malaria, and none for tuberculosis. Some evidence is already available, and some genes have already been identified, such as CYP2D6 for primaquine treatment and NAT2 for isoniazid. However, some barriers to the implementation are the lack of evidence due to the few studies on the diseases themselves and the admixture of the most affected populations, which must be considered, given the genetic differentiation of these populations. Conclusions: PGx tests such as abacavir are already implemented in some places, and efavirenz/atazanavir is ready to implement if this medication is used. Other gene–drug associations were found but still do not present a clear recommendation. We call attention to the need to generate more evidence for testing treatments for other neglected diseases, such as malaria and tuberculosis, given their epidemiological importance and for the public health of less favored populations. Full article

(This article belongs to the Special Issue Pharmacogenomics in Infectious Diseases)

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17 pages, 5065 KiB

Open AccessArticle

Genome-Wide microRNA Expression Profiling in Human Spermatozoa and Its Relation to Sperm Quality

by Nino-Guy Cassuto, Florence Boitrelle, Hakima Mouik, Lionel Larue, Gwenola Keromnes, Nathalie Lédée, Laura Part-Ellenberg, Geraldine Dray, Léa Ruoso, Alexandre Rouen, John De Vos and Said Assou

Genes 2025, 16(1), 53; https://doi.org/10.3390/genes16010053 - 4 Jan 2025

Viewed by 670

Abstract

Background: Sperm samples are separated into bad and good quality samples in function of their phenotype, but this does not indicate their genetic quality. Methods: Here, we used GeneChip miRNA arrays to analyze microRNA expression in ten semen samples selected based on high-magnification [...] Read more.

Background: Sperm samples are separated into bad and good quality samples in function of their phenotype, but this does not indicate their genetic quality. Methods: Here, we used GeneChip miRNA arrays to analyze microRNA expression in ten semen samples selected based on high-magnification morphology (score 6 vs. score 0) to identify miRNAs linked to sperm phenotype. Results: We found 86 upregulated and 21 downregulated miRNAs in good-quality sperm (score 6) compared with bad-quality sperm samples (score 0) (fold change > 2 and p-value < 0.05). MiR-34 (FC × 30, p = 8.43 × 10⁻⁸), miR-30 (FC × 12, p = 3.75 × 10⁻⁶), miR-122 (FC × 8, p = 0.0031), miR-20 (FC × 5.6, p = 0.0223), miR-182 (FC × 4.83, p = 0.0008) and miR-191 (FC × 4, p = 1.61 × 10⁻⁶) were among these upregulated miRNAs. In silico prediction algorithms predicted that miRNAs upregulated in good-quality sperm targeted 910 genes involved in key biological functions of spermatozoa, such as cell death and survival, cellular movement, molecular transport, response to stimuli, metabolism, and the regulation of oxidative stress. Genes deregulated in bad-quality sperm were involved in cell growth and proliferation. Conclusions: This study reveals that miRNA profiling may provide potential biomarkers of sperm quality. Full article

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

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Differences in the global miRNA expression profiles of S6 and S0 sperm samples. (A). Unsupervised 3D PCA representing the miRNA expression patterns of S6 spermatozoa (n = 5 samples) and S0 spermatozoa (n = 5 samples). Each sample was analyzed using the GeneChip® miRNA 4.0 Array. Red dots, S6 samples; blue dots, S0 samples. (B). Hierarchical clustering of the samples using the differentially expressed miRNAs with the highest variation. S6 and S0 samples (n = 5/each group) are clustered in two distinct groups. (C). Heat map of the S6 and S0 miRNA signatures based on the 107 miRNAs that are differentially expressed between S6 and S0 samples. Each column corresponds to a specific miRNA, and each row represents a sperm sample. The color scale reflects the relative miRNA expression levels, with red indicating higher expression and blue indicating lower expression. (D). Violin plots showing the expression of the top 10 upregulated miRNAs in S6 samples based on the TAC analysis of the microarray data. S6: good quality samples, S0: bad quality samples. Full article ">Figure 2
Analysis of GO terms associated with S6-miRNA targets and their functions. (A). Analysis of significantly represented GO terms. Pathway enrichment analyses were carried out using the human gene names of S6-miRNA targets. The size of the blue dots reflects the degree of enrichment, with larger dots representing more significant p-values. (B). GSEA was conducted using the S6-miRNA targets. The heat map illustrates the clustering of genes within the leading-edge subsets, emphasizing the dynamic expression of genes associated with programmed cell death regulation, phosphorylation, positive regulation of cell proliferation, and metabolic processes. Genes are shown on the vertical bars colored from deep blue (top rank) to blank (lowest rank). (C). Bubble plot of the overlapping canonical pathways associated with S6-miRNA targets. The circle size reflects the number of genes involved in the pathway. The canonical pathways were categorized into various types based on the IPA database. Full article ">Figure 3
Top-ranked functional networks of the S6-miRNA target genes. Top networks identified by IPA of S6-miRNA target genes related cell growth and proliferation, cell cycle regulation, DNA replication and repair, system development and function, tissue morphology, reproductive system disorders, cell morphology, cellular assembly and organization, cellular function and maintenance, cell death and survival, and developmental disorders. Green nodes represent genes regulated by S6-miRNAs. Dashed lines represent indirect relationships, while solid lines indicate direct molecular interactions. Within each network, the edge types are defined as follows: a line without an arrowhead signifies binding only, a line ending with a vertical bar represents inhibition, and a line with an arrowhead indicates an “acts on” relationship. *: indicate that several gene identifiers in the dataset file correspond to a single gene in the Global Molecular Network. Full article ">Figure 4
Networks of the S6-miRNA target genes. The IPA tool was used to generate the networks based on the predicted miRNA–mRNA interactions. Pink nodes represent the miRNAs upregulated in S6 samples and green nodes represent the genes targeted by S6-miRNAs. Solid lines represent direct interactions and dashed lines indirect interactions. *: indicate that several gene identifiers in the dataset file correspond to a single gene in the Global Molecular Network. Full article ">Figure 5
The promoters of the predicted S6-miRNA target genes are not differentially methylated. Integrative Genome Viewer snapshots illustrating the methylation levels at individual CpG sites (0–100%) across the examined genes. Each promoter region (red arrow) overlaps with a CpG island (green box). Full article ">Figure 6
Enrichment of S6-miRNA targets in critical signaling pathways and their expression in testes. (A). Pathway analysis (KEGG pathway) using the Pathview server (<a href="https://pathview.uncc.edu/" target="_blank">https://pathview.uncc.edu/</a> (accessed on 17 June 2024)). Highlighted genes are pathway components identified as targets of S6 miRNAs. (B). Expression profile of candidate genes in various human tissues. Expression levels (in Log2 RPKM) of PDGFA, PDGFRA, GRB2, MECP2, MAP2K1, ARHGDIA, and MET in 30 tissues from GTEx. For each gene, the colored circle corresponding to each tissue represents the RPKM value averaged across all samples within that tissue. RPKM stands for reads per kilobase of transcript per million mapped reads. Full article ">

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