Integrated proteomic profiling of aqueous humor reveals CDC42/RHOA-mediated pathogenic mechanisms in nAMD and PDR.

Objective: To explore the role and underlying mechanisms of transient receptor potential vanilloid 4 (TRPV4) in pressure overload-induced myocardial fibrosis in fetal tetralogy of Fallot (TOF). Methods: Fetal cardiac samples diagnosed by prenatal echocardiography or postmortem examination were retrospectively collected from Qingdao University Affiliated Women and Children's Hospital between May 2022 and July 2024, including 4 cases in the Tetralogy of Fallot (TOF) group and 4 cases in the structurally normal heart control group. Histological analysis of myocardial hypertrophy, fibrosis, and collagen content was performed on collected right ventricular wall tissues using hematoxylin-eosin (HE), Masson＇s trichrome, and Sirius red staining. Immunohistochemistry was used to detect the expression levels of TRPV4, Ras homolog gene family member A (RhoA), and Rho-associated coiled-coil containing protein kinase 1 (ROCK1) in myocardial tissues, while Western blot was applied to measure the expression of Collagen Ⅰ, Collagen Ⅲ, and α-smooth muscle actin (α-SMA). In vitro, human umbilical vein endothelial cells (HUVEC) at passages 3 to 6 were seeded on hydrogel-coated plates with different stiffnesses (8 kPa, 50 kPa) and divided into 8 kPa, 50 kPa, and 50 kPa+TRPV4 inhibitor (pretreated with a TRPV4 antagonist) groups. Small interfering RNA targeting TRPV4 (siTRPV4) and negative control siRNA (siNC) were transfected into HUVECs to establish TRPV4-knockdown models. These cells were then seeded on hydrogel plates of varying stiffness and divided into 8 kPa, 50 kPa, siNC, and siTRPV4 groups. Western blot was used to assess the expression levels of TRPV4, RhoA, ROCK1, Collagen Ⅰ, Collagen Ⅲ, α-SMA, CD31, vascular endothelial cadherin (VE-cadherin), and endothelial-mesenchymal transition (EndMT)-related regulatory proteins (p-Smad2 and the transcription factor Slug). Results: HE staining revealed cardiomyocyte hypertrophy in the right ventricular wall of the TOF group compared with the control group, accompanied by varying degrees of myocardial disarray. Masson＇s trichrome staining indicated a higher percentage of fibrotic area in the right ventricular myocardium of the TOF group, and Sirius red staining showed a larger collagen deposition area (all P<0.05). Compared with the control group, the TOF group exhibited significantly higher protein expression of Collagen Ⅰ, Collagen Ⅲ, and α-SMA, along with enhanced positive signal intensity for TRPV4, RhoA, and ROCK1 (all P<0.05). In vitro experiments, compared with the 50 kPa group, both the 8 kPa group and the 50 kPa+TRPV4 inhibitor group had lower expression levels of Collagen Ⅰ, Collagen Ⅲ, α-SMA, p-Smad2, Slug, TRPV4, RhoA, and ROCK1, while expression levels of CD31 and VE-cadherin were higher (all P<0.05). Similarly, the siTRPV4 group showed lower expression of Collagen Ⅰ, Collagen Ⅲ, α-SMA, TRPV4, RhoA, ROCK1, Slug and p-Smad2, and higher expression of CD31 and VE-cadherin compared with the 50 kPa group (all P<0.05). In contrast, no significant differences in the expression of these proteins were observed between the siNC group and the 50 kPa group (all P>0.05). Conclusions: The right ventricle myocardium of TOF fetuses exhibits evident myocardial fibrosis. Mechanical stimulation induced by high-pressure load activates TRPV4, which may mediate EndMT in cardiac vascular endothelial cells through regulation of the RhoA/ROCK1 signaling pathway. This mechanism may represent one of the primary pathological contributors to myocardial fibrosis development.

The majority of cases of autosomal-dominant retinitis pigmentosa (adRP) are associated with rhodopsin (RHO) variants. More than 290 pathogenic variants responsible for 25%-30% of adRP cases have been identified to date. This retrospective report focuses on RHO and RP cases in the Brazilian population. Patients with molecular confirmation of pathogenic variants in the RHO gene were included. Their clinical and genetic data were analyzed. Segregation analyses were included where possible. Cases were classified as generalized RP or sector RP according to fundus examinations and imaging data. The medical records of 43 patients from 34 families with RHO-associated RP were reviewed. Twenty-two disease-causing variants of the RHO gene and four previously unreported variants (c.317G>T; c.937-2A>T, c.272_283del, and c.530+1G>C) were identified. The majority of cases involved missense variants. The most prevalent variant was c.551A>G, p.(Gln184Arg), which was identified in seven patients (21%) from four families. One patient presented with the splice donor variant c.530+1G>C in the homozygous state, which was classified as pathogenic. Thirty-two patients presented with a generalized RP phenotype, and six patients were diagnosed with sector RP. This study provides information on the clinical and genetic features of RHO-associated RP in the Brazilian population, expanding the spectrum of RHO gene disease-causing variant frequencies.

RhoA/ROCK1 aggravates transverse aortic constriction-induced atrial fibrillation by enhancing NF-κBp65/CCL2 signaling pathway.

Subchondral bone is a dynamic tissue maintained by bone remodeling and responded rapidly to mechanical loading, which is strongly associated with cartilage degradation in osteoarthritis (OA), but the underlying mechanisms of which is still blurring. Experimental OA mice models were generated by mechanical overload with 60 cycles of 14N axial compressive loads twice a week or anterior cruciate ligament transection surgery. FSCN1 expression was evaluated in subchondral bone from experimental OA mice and human OA. Mice with FSCN1 conditional knockout in pre-osteoblasts were generated, and adeno-associated virus expressing FSCN1 was injected intra-articularly in mice. Therapeutic efficacy of FSCN1 inhibitor NP-G2-044 was determined in OA mice. Increased FSCN1 expression was positively associated with osteogenesis and subchondral bone sclerosis induced by mechanical loading. Deletion of FSCN1 in pre-osteoblasts delays osteogenesis and prevents abnormal subchondral bone sclerosis, whereas overexpression of FSCN1 exacerbates this. Under the stimulation of mechanical stress, Ras homologue gene family member A in osteoblasts competitively binds with protein kinase C to FSCN1, thereby inhibiting FSCN1 phosphorylation and promoting cytoskeleton formation, thereby activating Hippo/YAP signaling to increase metabolic activity of osteoblasts. These abnormal osteoblasts secrete more osteogenesis proteins, including osteopontin, leading to subchondral bone sclerosis and cartilage erosion, thus aggravating the progression of OA. Furthermore, we confirmed that the inhibitor of FSCN1, NP-G2-044 effectively attenuates subchondral bone sclerosis and OA progression in mice. This study suggests that FSCN1 is a key factor in the relationship between mechanical stress, actin cytoskeleton dynamic, subchondral bone sclerosis and OA pathology. Targeting FSCN1 represents a promising pharmacological approach for OA therapy.

To observe the therapeutic effects of growth associated protein-43 (GAP 43) gene modified rat bone marrow mesenchymal stem cells (BMSCs) on experimental retinal degeneration. A total of 80 male Royal College of Surgeons (RCS) rats were divided into three groups at postnatal 21 (P21) randomly. A cell suspension of 5 × 104 BMSCs modified with GAP-43 in 2µl PBS was injected into the subretinal space of BMSCs + GAP-43 group rats, BMSCs group rats received5 × 104 BMSCs in 2µl PBS and PBS group rats received 2µl PBS. Photoreceptor necrosis and apoptosis was assessed by HE staining and Terminal-deoxynucleotidyl Transferase Mediated Nick End Labeling (TUNEL) detection. The expression of GAP-43, Glutamine synthetase (GS), rhodopsin (RHO), Caspase-8 and Caspase-9 was analyzed by immunofluorescence, western blot. Transplantation of GAP-43-modified BMSCs into the subretinal cavity of RCS rats revealed that: retinal GAP-43 expression was significantly elevated (P < 0.05); Rhodopsin (Rho) expression, a photoreceptor marker, was upregulated 1.5-fold (P < 0.05), and the thickness of the outer nuclear layer increased to 79.78 ± 6.83μm (control 29.92 ± 4.17μm, P < 0.01); The apoptosis rate decreased to 41.9% (control 63.8%, P < 0.01), and Caspase-8/9 activation was inhibited; Müller cell proliferation was reduced (GS decreased, P < 0.05). suggesting that GAP-43-modified BMSCs delayed retinal degeneration through anti-apoptosis and pro-photoreceptor survival. The results suggest that GAP-43 gene modified BMSCs has the therapeutic effect on the early stage of retinal degeneration, which might make more photoreceptors preserved.

Rod photoreceptors are essential for vision under dim light conditions and are highly vulnerable in retinal degenerative diseases. Here, we demonstrate that both human and rodent rods undergo a minute and rapid contraction of their outer segments upon photoisomerization, the first step of phototransduction. The contraction is explained as an electromechanical manifestation of the rod early receptor potential generated in the disk membranes, which is challenging to access in electrophysiology. The in vivo optical imaging of light-evoked electrical activity in rodent rods was facilitated by an ultrahigh-resolution point-scan optical coherence tomography (OCT) system, combined with an unsupervised learning approach to separate the light-evoked response of the rod outer segment tips from the retinal pigment epithelium-Bruch’s membrane complex. In humans, an adaptive optics line-scan OCT facilitated high-speed recordings in rods. The non-invasive in vivo optical imaging of rhodopsin activation extends the diagnostic capability of optoretinography, and may facilitate personalized, objective assessment of rod dysfunction and visual cycle impairment in inherited and age-related macular degeneration.

Ocular delivery of different valosin-containing protein (VCP) inhibitory formulations prevents retinal degeneration in rho∆I255 mice.

Background/Objectives: Targeted gene sequencing (TGS) for Comprehensive Genomic Profiling (CGP) use in sarcomas has recently increased in clinical practice. We report on TGS real-world data over a period of 3 years (2022-2025) at the IRCCS Istituto Ortopedico Rizzoli, with the aim of identifying potential actionable targets and providing therapeutic indications for advanced sarcoma patients. Methods: We analyzed 22 advanced sarcoma patients by using the VariantPlex Pan Solid Tumor kit panel, including 185 genes. In nine cases, saliva samples for germinal DNA analysis were available. Sequencing was performed on the NextSeq-500 Platform and analyzed with Archer Analysis software. The Cancer Genome Interpreter and OncoKB Database tools were used to find potential actionable targets. Results: We found the most frequent genetic variants, including missense, deletion, duplication, and delins, in the NOTCH4, AR, BARD1, MUC16, and ROS1 genes. Copy Number alterations affected the CDKN2A, CDKN2B, TP53, RHOA, MYC, CCND3, and DDR2 genes mainly in osteosarcoma samples. In four patients, longitudinal analyses of subsequent lesions showed the maintenance of most genomic alterations and enrichment in missense or splice variants in PMS2, SMARCA4, ARID1A, AKT1, BMPR1A, and PTEN, indicating the occurrence of tumor evolution. Germline variants subtraction identified the specific somatic tumor mutations. Advantages and disadvantages of our approach were considered in order to refine the analysis setting and better select possible actionable targets. Conclusions: Early access to genomic analyses, routine germline assessment, and broad gene panels would help in identifying possible targeted drugs with sufficient evidence of activity beneficial to each patient. In the clinical management of advanced sarcoma patients, when analyzing cost-effectiveness and sustainability, the role of the Molecular Tumor Board in the governance of the complexity introduced by mutational oncology should be considered.

Rhodopsin is a photoreceptor protein found in the vertebrate retina used as a landmark for vision evolution studies at the molecular level. Here, we examined the biochemical and functional performance of modern rhodopsin from three different mammal species— bovine, murine and human—to analyze their visual pigment evolutionary relationships. We selected these species for their relevance in vision research, their different position on the phylogenetic tree and their diverse ethology regarding nocturnal (mouse) and diurnal (bovine and human) life. We report on the important role of the amino acid at position 290 as a key player in the active rhodopsin conformation stability. Our spectroscopic analysis shows that the retinal release process for mouse rhodopsin (L290) is significantly slower, meaning a more stable and durable active state, compared to the human and bovine cases (I290). This finding is supported by the faster retinal release rate observed in the L290I mutant mouse rhodopsin, where the nocturnal mutated pigment behaved like diurnal rhodopsin. The result suggests a potential link between the amino acid at this position and the activity pattern (nocturnal or diurnal). This association was also observed when comparing the sequences of 79 mammal species at position 290, and better appreciated in more specialized primate and rodent orders. Moreover, we propose an evolutionary mechanism in rhodopsin specialization for diurnal and nocturnal life, implying a compromise between the prevalence of damage protection under bright light in nocturnal therian mammals (L290) and dark adaptation under dim light in diurnal therian mammals (I290).Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-29872-9.

Aim: This study aims to investigate the expression of the Ankyrin Repeat Domain 6 (ANKRD6) gene in Colon Adenocarcinoma (COAD) and its regulatory role in key signaling pathways, evaluating its clinical value as a potential therapeutic target. Methods: To investigate the functional role of ANKRD6 in colon adenocarcinoma (COAD), we systematically analyzed its prognostic relevance, epigenetic regulation, and association with tumor stemness using publicly available TCGA (The Cancer Genome Atlas)-COAD data. Gene set variation analysis (GSVA) was applied to identify signaling pathways potentially modulated by ANKRD6. Least Absolute Shrinkage and Selection Operator (LASSO) regression was employed to identify key genes within the Wnt signaling pathway that are significantly associated with COAD patient survival. Immunohistochemical staining was performed to confirm ANKRD6 protein expression in COAD tissues. To investigate functional consequences, ANKRD6 was knocked down using small interfering RNAs, and subsequent quantitative real-time polymerase chain reaction and Western blot assays were applied to measure alterations in representative Wnt pathway-related genes and proteins. Results: ANKRD6 expression was significantly associated with DNA methylation patterns, RNA modification regulators, and tumor stemness features in COAD. These findings suggest that epigenetic and post-transcriptional mechanisms may underlie the regulatory role of ANKRD6 in COAD pathogenesis. GSVA analysis revealed that ANKRD6 significantly influences and negatively regulates Wnt signaling pathway activity. LASSO regression analysis highlighted 25 Wnt pathway genes relevant to COAD patient survival, with ANKRD6 expression significantly correlated with multiple targets. Furthermore, immunohistochemical staining confirmed that ANKRD6 protein was markedly upregulated in COAD tissues. Upon ANKRD6 knockdown, the messenger RNA levels of WNT7A (Wnt Family Member 7A), JNK (Mitogen-Activated Protein Kinase 8), and RHOA (Ras Homolog Family Member A), as well as WNT7A protein levels, showed a significant decrease in the Wnt signaling pathway. Conclusions: ANKRD6 plays a critical role in the development and progression of COAD by regulating the Wnt signaling pathway. It holds potential as a therapeutic target, warranting further investigation into its specific regulatory mechanisms and clinical applications.

Cerium oxide nanoparticles (CeO2 NPs), a few nanometers in diameter, exhibit strong antioxidant and anti-inflammatory activity and are considered promising candidates for the treatment of ocular diseases. This study investigated the impact of CeO2 NPs, both in their naked form, and formulated as eye drops, on the bacteria inhabiting the ocular surface. The microbiome from healthy eyes was characterized through 16S rRNA amplicon sequencing. The dominant bacterial genera included Staphylococcus, with lower abundances of Corynebacterium and Streptococcus, varying among the samples. Eye bacteria were isolated and exposed to both forms of CeO2 NPs to investigate cell growth, viability, and gene expression. Neither naked CeO2 NPs nor eye drop formulation affected the growth of Staphylococcus aureus and Staphylococcus epidermidis. In S. aureus, CeO2 caused down-regulation of rho (naked CeO2 NPs) and recA (eye drop form) genes related to the termination of transcription and DNA damage response, respectively. In S. epidermidis, the expression of lexA, recA, ftsZ, rho and icaC remained comparable between treated and control samples, while DNA damage response genes lexA and recA were up-regulated in C. amycolatum following exposure to CeO2 eye drops. These gene expression patterns revealed subtle changes in specific bacteria, indicating a short-term adaptive response to eye drop formulations. Overall, these results suggest that CeO2 NP eye drops have a minimal impact on the isolated bacterial strains. Nonetheless, comprehensive in vivo and clinical studies are necessary to validate these findings, given the inherent limitations of in vitro assays.

Mutations in rhodopsin (RHO) cause autosomal-dominant RP, which has multiple clinical subclasses, including class B1 ("sector") RP in which the asymmetric retinal degeneration (RD) suggests an environmental influence. The pathogenic mechanisms of most class B1 mutations are uncharacterized. We generated new animal models of RHO-associated RP to examine RHO expression, localization, glycosylation, and the influence of light and chromophore binding on RD. We generated transgenic Xenopus laevis expressing wild-type or mutant human RHO transgenes. Confocal images were used to evaluate RD and trafficking. Immunoassays were used to quantify RD and investigate RHO glycosylation. We created X. laevis models of 26 different forms of RHO-associated RP. Most mutations caused RD, with the exception of those at residues F45, G101, and R135. Many variants did not alter RHO localization. Multiple class B1-associated RHO mutants induced light-dependent RD, suggesting light is the environmental influence associated with the class B1 phenotype. However, the degeneration associated with two partially endoplasmic reticulum-retained class B1 mutants (S22R and D190G) was not mitigated by dark rearing. P23H and S176F constituted a distinct subclass associated with inner segment retention and proteolytic cleavage. Many RHO mutations do not substantially alter RHO localization or glycosylation. The exceptions we identified are P23H and S176F, which dramatically mislocalize, and constitute a distinct category of proteolytically cleaved misfolding variants. L31Q and T58R induce RD by mechanisms similar to glycosylation-deficient variants, despite the lack of glycosylation defects. Intermediate phenotypes indicate at least one previously undescribed mechanism for class B1 RP pathogenesis.

Lighting is routinely specified only by its impact on the three cone photoreceptors via the correlated color temperature (CCT), ignoring the visual and non-visual contributions of the melanopsin photoreceptors. Disentangling the behavioral effects of the CCT from those of themelanopsin excitation is complex but necessary to understand melanopsin's effects and to inform the design of new lighting spectra for the built environment. Melanopsin photoreception is important for driving many visual and non-visual functions in humans, including circadian rhythms, mood, attention, and arousal. Here, we introduce a methodology using a widely available LED source (Philips Hue Play, Signify N.V.) to decouple the effects of melanopsin from those of cone photoreceptors. We present a computational algorithm for producing two ambient illuminations with different melanopsin and rhodopsin activation levels, whilst maintaining the same cone excitations, CCT and visual appearance (i.e., the two lighting conditions are cone metamers); this simple and inexpensive method removes the major confounding factor present in approaches that alter the melanopsin excitation of a light by exchanging the wavelength, color, or CCT. The method may find applications in behavioral experiments, including for clinical trials.

To repair excised portions of the nose in non-melanoma skin cancer, surgeons have begun to use tissue-engineered nasal cartilage autografts. While promising, this approach has been hindered by significant inter-donor variability of cartilage quality within the autografts. For this approach to become clinically viable, there is a need to pre-select nasal chondrocytes with the capacity to create high-quality cartilage for autograft engineering. Recently, using RNA sequencing of clonal nasal chondrocyte populations, we have discovered G protein-coupled receptor 56 (aka ADGRG1 ) expression to be positively correlated with glycosaminoglycan per DNA (cellular content) and SOX9 expression, both are metrics of chondrogenic capacity. This correlation suggests G protein-coupled receptor 56 holds promise to be a marker of nasal chondrocytes with the capacity to produce high quality cartilage. G protein-coupled receptor 56 has been extensively studied in fields such as neural development, hematopoiesis, and cancer but is unreported within the cartilage biology field. This narrative review examines the molecular, cellular and physiological literature of G protein-coupled receptor 56, including some of its known ligands: collagen III, transglutaminase 2, testosterone, and signaling pathways: Ras homolog gene family, member A/Rho-associated protein kinase, phosphoinositide 3-kinase/AKT/mechanistic target of rapamycin, Nuclear Factor of Activated T-cells, cyclic adenosine monophosphate/protein kinase A. We then integrate the literature of these ligands and signaling pathways from the cartilage biology field to hypothesize the specific cellular and physiological role of G protein-coupled receptor 56 within cartilage. Ultimately, this review highlights the promise of G protein-coupled receptor 56 as a cell marker for high-quality cartilage production, we emphasize the need for further empirical investigation of G protein-coupled receptor 56 in the cartilage.

Retinal organoids mirror CRISPR-Cas9 gene editing efficiency observed in vivo

Congenital stationary night blindness (CSNB) is a rare group of genetically inherited retinal disorders that are present from birth and do not show progressive characteristics. It is typically accompanied by symptoms such as night blindness, photophobia, myopia, nystagmus, and strabismus. CSNB is classified into subtypes based on different electrophysiological findings and inheritance patterns. Cases with a normal fundus appearance are categorized into Riggs type and Schubert-Bornschein type (which includes complete and incomplete subtypes) based on electroretinographic findings, while abnormal fundus appearance characterizes forms such as fundus albipunctatus (FA) and Oguchi disease. The genetic variations responsible for CSNB differ according to its subtypes. Riggs type CSNB is commonly associated with mutations in the PDE6B, RHO, GNAT1, and SLC24A1 genes. Complete CSNB is linked to mutations in GRM6, TRPM1, GPR179, LRIT3, and NYX genes, whereas incomplete CSNB involves mutations in CABP4, CACNA2D4, and CACNA1F. In FA, which presents with an abnormal fundus, mutations are often found in the RDH5, RLBP1, RPE65, and LRAT genes. Oguchi disease is associated with mutations in the SAG and GRK1 genes. Most of these genes are related to retinal photoreceptors, bipolar cells, or proteins involved in the retinoid cycle and play roles in signal transmission or the visual cycle.

Objective To investigate the effect and mechanism of baicalin on blood lipid metabolism and immune function in rats with gestational diabetes mellitus (GDM). Methods Female rats fed with high-fat and high-sugar diet and male rats fed with ordinary diet were caged together to prepare pregnant rats, and the GDM rat model was established by intraperitoneal injection of streptozotocin (35 mg/kg). GDM rats were randomly divided into a model group, a fasudil (FA) (RhoA/RocK inhibitor) group (10 mg/kg), low-dose (100 mg/kg) and high-dose (200 mg/kg) baicalin groups, and a high-dose baicalin combined with LPA (RhoA/RocK activator) group (200 mg/kg baicalin+1 mg/kg LPA ), with 12 rats in each group. Another 12 pregnant rats fed with high-fat and high-sugar diet were selected as the control group. After 2 weeks of corresponding drug intervention in each group, the level of fasting blood glucose (FBG) was detected by blood glucose meter. The level of fasting insulin (FINS) in serum was detected by ELISA, and the insulin resistance index (HOMA-IR) was calculated. The levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total cholesterol (TC), triglyceride (TG), low density lipoprotein cholesterol (LDL-C), high density lipoprotein cholesterol (HDL-C) in serum, and the levels of immunomodulator tumor necrosis factor α (TNF-α), interleukin 6 (IL-6), and IL-10 in peripheral blood were detected by the kit. The histopathological changes of liver were observed by HE staining. The proportion of T lymphocyte subsets in peripheral blood was detected by flow cytometry. The mRNA and protein expressions of Ras homolog gene family member A (RhoA), Rho associated coiled-coil forming protein kinase 1 (ROCK1), and ROCK2 in liver tissue were detected by real-time quantitative PCR and Western blot. Results Compared with the control group, the levels of FBG, FINS, HOMA-IR, ALT, AST, TG, TC, and LDL-C in serum, the levels of TNF-α, IL-6, the percentage of CD8+T cell in peripheral blood, and the mRNA and protein expression of RhoA, ROCK1, and ROCK2 in liver tissue in the model group were higher; the level of HDL-C in serum, the percentage of IL-10 levels, CD3+T cells, CD4+T cell, and CD4+T/CD8+T ratio in peripheral blood were lower. Compared with the model group, the levels of FBG, FINS, HOMA-IR, ALT, AST, TG, TC, and LDL-C in serum, the levels of TNF-α, IL-6, the percentage of CD8+T cell in peripheral blood, and the mRNA and protein expression of RhoA, ROCK1, and ROCK2 in liver tissue in the the FA group and low-dose and high-dose baicalin groups were lower; the level of HDL-C in serum, IL-10 level, the percentage of CD3+T cells, CD4+T cell, and CD4+T/CD8+T ratio in peripheral blood were higher. LPA could obviously weaken the improvement effects of baicalin on blood lipid metabolism and immune function in GDM rats. Conclusion Baicalin may improve blood lipid metabolism and immune function in GDM rats by inhibiting the RhoA/ROCK pathway.

Background: Foetal structural abnormalities can be detected in approximately 3% of all pregnancies and frequently remain without a genetic diagnosis. This study aims to apply an integrated approach with the final goal of providing a molecular diagnosis in the challenging Italian setting of early termination of pregnancy. Methods: In a cohort of 86 foetuses, post-mortem dysmorphological examination, radiological assessments, and molecular autopsy through Whole-Exome Sequencing—WES—analysis were performed. Results: Forty-two foetuses were phenotypically classified as presenting a single major malformation (i.e., central nervous system, skeletal, urogenital, or cardiac anomalies, or fluid accumulation), while 44 foetuses presented multiple malformations and/or dysmorphic features. Overall, WES provided a diagnostic yield of 26.7%; additionally, seven Variants of Uncertain Significance (VUS) potentially liked to the foetal phenotype were identified. The highest detection rate was achieved for foetuses presenting a single major urogenital (50%) or skeletal (42.9%) malformation, followed by foetuses presenting multiple malformations (27.3%). Peculiar results of particular interest were (1) the identification of two splicing variants (within the INPPL1 and RHOA genes), functionally characterised through minigene assay, which contributed to evaluate their pathogenicity, and (2) the identification of a novel de novo missense ZNF292 variant (NM_015021.3:c.6325A>C p.(Ser2109Arg)) in a foetus affected by corpus callosum hypoplasia. The ZNF292 gene is associated with the Intellectual developmental disorder, autosomal dominant 64 and this finding represents the first report of prenatally detected anomalies of the central nervous system in a foetus carrying a ZNF292 variant. Conclusions: This study underlines the diagnostic utility of an integrated approach to achieve a precise genetic diagnosis for structural foetal abnormalities, thus providing families with precise recurrence risk estimations and detailed options about future pregnancies. Additionally, a systematic implementation of this strategy could be crucial to better characterise new variants and discover new genes involved in embryonic and foetal development.

Autosomal dominant retinitis pigmentosa: An extended family report of the Asp-190-Tyr variant.