COL1A1 Gene Research Articles

Abstract 4134967: Pericardial Adipose Tissue Hypertrophy Contributes to Heart Failure via β3 Adrenergic Signaling-Mediated Activation of the TGFβ1 Pathway

Background: The heart is surrounded by pericardial adipose tissue (PeAT). An increase in PeAT volume has been associated with the development of heart failure; however, the precise molecular mechanisms by which hypertrophic PeAT contributes to heart failure remain to be elucidated. Aims: To clarify the involvement of PeAT hypertrophy in the development of heart failure. Methods: Male C57BL/6 mice (8-10 weeks old) were fed a high-fat diet (HFD) for 8 weeks to induce PeAT hypertrophy. Cardiac pressure overload was induced by transverse aortic constriction (TAC) with a 26-gauge needle. Results: PeAT volume significantly increased in HFD-fed mice compared to those on a normal diet (ND) (25.28 ± 4.1 mg vs. 9.35 ± 0.73 mg, p<0.01). Eight weeks after TAC, HFD-fed mice (TAC+HFD) exhibited more pronounced cardiac dysfunction (fractional shortening: 45.21 ± 2.21% vs. 55.99 ± 1.91%, p=0.002, left ventricular mass: 130.3 ± 7.80% vs. 106.8 ± 3.77%, p=0.0154), upregulation of cardiac fibrosis-related genes ( Col1a1 , Col3a1 ), and greater fibrotic area in the left ventricle compared to ND-fed TAC mice (TAC+ND). Simultaneously, the TGFβ/Smad pathway was activated in the left ventricles of TAC+HFD mice. Notably, surgical removal of hypertrophic PeAT improved cardiac function in TAC+HFD mice, while transplantation of PeAT from HFD-fed mice into TAC+ND mice worsened cardiac function. In TAC+HFD mice, local noradrenaline concentration in PeAT decreased, along with reduced activation of hormone-sensitive lipase, a downstream target of β3 adrenergic receptor signaling. Local free fatty acid concentrations also decreased in the PeAT of HFD-fed mice. In the RAW 264.7 macrophage cell line, a decrease in palmitic acid, a major fatty acid, led to increased expression of TGFβ1. Conclusions: These results suggest that HFD-induced PeAT hypertrophy suppresses β3 adrenergic receptor signaling and free fatty acid release, increasing TGFβ1 expression in the macrophages and resulting in cardiac fibrosis and dysfunction. Our findings provide new insights into the pathogenesis of PeAT hypertrophy in heart failure development.

PATH-41. PRIMARY BRAIN SARCOMA WITH DROP METASTASES AND LEPTOMENINGEAL DISEASE

Abstract INTRODUCTION Primary brain sarcoma is extremely rare and has a very poor prognosis. Sarcomas are a highly diverse group, with over 100 different subtypes identified in the recent WHO classification. Case: In this case, we describe a 51-year-old man diagnosed with primary right frontal poorly differentiated sarcoma. He experienced cognitive changes for weeks. Brain MRI revealed a lobulated mass with peripheral enhancement measuring 67 mm by 61 mm by 52 mm. He underwent gross total resection, and pathology indicated high-grade poorly differentiated sarcomatous lesions. Further analysis at Memorial Sloan Kettering and NIH failed to match any DNA methylation class. Next generation sequence (NGS) testing revealed mutation of NF2, CDKN2A, COL3A1, CD33, and PIK3CA genes, increased PD-L1 mRNA, and PD-L1 expression, greater than 50%. The conclusive diagnosis was a PD-L1 high poorly differentiated sarcoma, not otherwise specified. Since no primary site was found elsewhere, planned to treat it as a primary brain sarcoma. Patient underwent focal radiation therapy with a plan to start immunotherapy combination of anti-CTLA4 and anti-PD1 therapy. In addition, an anti-VEGF therapy with pazopanib because of the efficacy of pazopanib in the setting of soft tissue sarcomas. The patient completed IMRT on 07/13/2023 and presented with a new onset of left leg weakness and urinary incontinence on 07/25/2024, further neuraxis imaging with MRI showed local and distant intracranial recurrence with drop metastasis at C1, C5-C6, and S2. The patient was started on IMRT to address drop metastasis, however further clinical decline led to treatment discontinuation. DISCUSSION There is no consensus on treatment guidelines on primary brain sarcomas given that they are exceedingly rare. DNA methylation and NGS provided very valuable information. We were unable to find any reported cases of primary brain sarcoma with drop metastases and leptomeningeal disease in the literature.

Potential of epicatechin as antioxidant and antiaging in UV-induced BJ cells by regulating COL1A1, FGF-2, GPX-1, and MMP-1 gene, protein levels, and apoptosis

Background Oxidative stress caused by exposure to ultraviolet (UV) light on the skin can damage deoxyribonucleic acid (DNA) and cause keratinocytes to undergo apoptosis. Endogenous antioxidants which play a role in trapping free radicals are also unable to overcome excess reactive oxygen species (ROS) in the body due to UV exposure, so exogenous antioxidants are needed. Polyphenolic compounds extracted from natural ingredients such as flavonoids, quercetin, and epicatechin have quite strong antioxidant activity. This is influenced by the chemical structure of these compounds which are rich in hydroxyl groups and aromatic groups. This structure allows the compound to become an electron donor so that it can neutralize free radicals. In vitro research was used to see the potential effectiveness of epicatechin as an antiaging and antioxidant. The study aims to confirm the potential of epicatechin as an antiaging by in vitro assay. Methods The viability test of epicatechin on human skin fibroblast (BJ) cells was carried out using the water-soluble tetrazolium (WST) assay. BJ cells were UV-induced as a cell model of premature aging. Epicatechin 6.25, 12.5, and 25 µg/mL were administered to UV-induced BJ cells. The gene expression of Collagen I Alpha 1 (COL1A1), matrix metalloproteinase-1 (MMP-1), fibroblast growth factor-2 (FGF-2), and glutathione peroxidase-1 (GPX-1) were analyzed by quantitative real-time polymerase chain reaction (qRT-PCR). Elastin (ELN), hyaluronidase (HAase), cyclooxigenase-2 (COX-2), 8-hydroxydeoxyguanosine (8-OhdG), and melatonin (MT) protein levels were analyzed by enzyme-linked immunosorbent assay (ELISA). The apoptosis of BJ cells was analyzed using flow cytometry. Results Treatment with epicatechin increased relative gene expression including COL1A1 (5.94), FGF-2 (8.34), and GPX-1 (8.09), and also decreased MMP-1 (2.90) relative gene expression compared to the UV-induced BJ cells. Epicatechin also increased levels of ELN (107.7 ng/mg protein) and MT (830 ng/mg protein) levels compared to the UV-induced BJ cells. Epicatechin treatment decreased levels of HAase (505.96 ng/mg protein), COX-2 (33.69 ng/mg protein), and 8-OHdG (97.87 ng/mg protein) compared to the UV-induced BJ cells. Epicatechin also succeeded in maintaining the percentage of live cells and reducing apoptosis, necrotic of UV-induced skin fibroblast cells. Conclusions Epicatechin has the potential to be an antiaging agent by in vitro assay.

Structural Variants in COL1A1 and COL1A2 in Osteogenesis Imperfecta.

Osteogenesis Imperfecta (OI) is a heterogeneous skeletal dysplasia characterized by bone fragility, skeletal deformities, and short stature. Most commonly, it is caused by autosomal dominant variants in the type I collagen genes, COL1A1 or COL1A2. Type I collagen is the main protein of the extracellular matrix in the skeleton and changes in its structure or quantity may lead to OI. 85%-90% of OI cases occur due to sequence variants in type I collagen genes, while OI caused by structural abnormalities in type I collagen genes is less common. In most cases, haploinsufficiency of type I collagen is associated with a milder OI phenotype. Large genomic deletions often involve several genes within the same chromosomal region, leading to microdeletion syndromes with OI features. Here, we report eight Swedish patients from five unrelated families with OI due to structural variants in the COL1A1 and COL1A2 genes. One patient with OI type III had a complex rearrangement with a deletion and duplication event in COL1A2, leading to reduced COL1A2 expression. Three other patients from two different families with OI type I had whole gene deletions involving COL1A1. In one family, three affected individuals with OI type I had a small intragenic deletion of exons 11-12 in COL1A2. One patient had a 2.1 Mb de novo deletion encompassing COL1A1 and DLX3 genes and features of OI and tricho-dento-osseous syndrome. Overall, this study highlights the importance of investigating gene dosage abnormalities in patients with OI and further delineates clinical and genetic variability of OI caused by structural variants in type I collagen genes.

A bizarre case of recurrent hemoptysis and pneumothorax in a young male

Vascular Ehlers–Danlos syndrome (vEDS) is a rare inherited disorder affecting approximately 1 in 150,000 individuals, caused by heterozygous mutations in the COL3A1 gene. Characterized by primary spontaneous pneumothorax and severe connective tissue fragility, vEDS remains poorly understood clinically, making diagnosis challenging. We present the case of a 20-year-old male with a history of mitral valve prolapse, recurrent hemoptysis, knee joint dislocations, and easy bruising. In February 2023, he was admitted to the emergency department following a seizure, syncope, and altered sensorium, which led to an extensive evaluation of his non-specific symptoms. Ultimately, he was diagnosed with vEDS.

Restored Collagen VI Microfilaments Network in the Extracellular Matrix of CRISPR-Edited Ullrich Congenital Muscular Dystrophy Fibroblasts

Collagen VI is an essential component of the extracellular matrix (ECM) composed by α1, α2 and α3 chains and encoded by COL6A1, COL6A2 and COL6A3 genes. Dominant negative pathogenic variants in COL6A genes result in defects in collagen VI protein and are implicated in the pathogenesis of muscular diseases, including Ullrich congenital muscular dystrophy (UCMD). Here, we designed a CRISPR genome editing strategy to tackle a dominant heterozygous deletion c.824_838del in exon 9 of the COL6A1 gene, causing a lack of secreted collagen VI in a patient’s dermal fibroblasts. The evaluation of efficiency and specificity of gene editing in treating patient’s fibroblasts revealed the 32% efficiency of editing the mutated allele but negligible editing of the wild-type allele. CRISPR-treated UCMD skin fibroblasts rescued the secretion of collagen VI in the ECM, which restored the ultrastructure of the collagen VI microfibril network. By using normal melanocytes as surrogates of muscle cells, we found that collagen VI secreted by the corrected patient’s skin fibroblasts recovered the anchorage to the cell surface, pointing to a functional improvement of the protein properties. These results support the application of the CRISPR editing approach to knock out COL6A1 mutated alleles and rescue the UCMD phenotype in patient-derived fibroblasts.

Osteoclast indices in osteogenesis imperfecta: systematic review and meta-analysis.

Osteogenesis imperfecta (OI) is a rare bone fragility disorder caused by mutations in genes encoding collagen type I or that affect its processing. Alterations in osteoclasts were suggested to contribute to OI pathophysiology. We aimed to systematically identify studies reporting measures of osteoclast formation and function in patients and mouse models of OI, to quantify OI-induced changes. The systematic search of Medline, Ovid, and Web of Science identified 798 unique studies. After screening, we included 23 studies for meta-analysis, reporting osteoclast parameters in 310 patients with OI of 9 different types and 16 studies reporting osteoclast parameters in 406 animals of 11 different OI mouse models. The standardized mean difference with 95% confidence interval (CI) was used as the effect size, and random-effects meta-analysis was performed. In patients with OI, collagen degradation markers were significantly higher compared with age-matched controls, with an effect size of 1.23 (CI: 0.36, 2.10]. Collagen degradation markers were the most elevated in the 3- to 7-year-old age group and in patients with more severe forms of OI. Bone histomorphometry demonstrated the trends for higher osteoclast numbers (1.16; CI: -0.22, 2.55) and osteoclast surface (0.43; CI: -0.63, 1.49), and significantly higher eroded surface (3.24; CI: 0.51, 5.96) compared with age-matched controls. In OI mice, meta-analysis demonstrated significant increases in collagen degradation markers (1.59; CI: 1.07, 2.11), in osteoclast numbers (0.94; CI: 0.50, 1.39), osteoclast surface (0.73; CI: 0.22, 1.23), and eroded surface (1.31; CI: 0.54, 2.08). The largest differences were in OI mice with the mutations in Col1a1 and Col1a2 genes. There were no differences between males and females in clinical or animal studies. Quantitative estimates of changes in osteoclast indices and their variance for patients with OI are important for planning future studies. We confirmed that similar changes are observed in mice with OI, supporting their translational utility.

A Novel Splice Site Variant in COL6A1 Causes Ullrich Congenital Muscular Dystrophy in a Consanguineous Malian Family

ABSTRACTBackgroundCongenital muscular dystrophies (CMDs) are diverse early‐onset conditions affecting skeletal muscle and connective tissue. This group includes collagen VI‐related dystrophies such as Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM), caused by mutations in the COL6A1, COL6A2 and COL6A3 genes. We report a consanguineous Malian family with three siblings affected by UCMD due to a novel homozygous splice site variant in the COL6A1 gene.MethodsAfter obtaining consent, three affected siblings and their relatives underwent physical examinations by specialists and laboratory tests where possible. DNA was extracted from peripheral blood for genetic testing, including Whole Exome Sequencing (WES). Putative variants were confirmed through Sanger Sequencing and assessed for pathogenicity using in silico tools.ResultsThe three siblings and their healthy parents, from a consanguineous marriage, presented with early‐onset progressive muscle weakness, walking difficulty, proximal motor deficits, severe muscle atrophy, hypotonia, skeletal deformities, joint hyperlaxity, ankyloses at the elbows and knees, keloid scars and dental crowding. No cardiac involvement was detected and creatine kinase (CK) levels were normal. All had low serum calcium levels, treated with oral supplements. Needle myography indicated myopathic patterns. WES identified a novel splice site variant in the first intron of COL6A1 (c.98‐1G>C), which segregated with the disease within the family. This variant is predicted to cause exon 2 skipping in COL6A1, with a high CADD score of 33 and Splice AI predicting it as deleterious.ConclusionWe identified a novel COL6A1 variant in a consanguineous family, highlighting the need for further studies in larger African cohorts to enhance genetic epidemiology and prepare for future therapeutic research.

A new paradigm of bone active risedronate-functionalized polycaprolactone/gelatin scaffold synergistically promotes osteogenic differentiation in rat bone marrow-derived mesenchymal stem cells for bone tissue engineering

Modern developments in bone tissue engineering focus on designing biomimetic materials that exhibit suitable mechanical and physiochemical properties. In this study, we present the development of a new biomaterial scaffold using a newly constructed polymer that contains bisphosphonate in combination with gelatin (GAT). To achieve scaffolding, a chemical grafting technique was used to develop risedronate (RS)-functionalized polycaprolactone (PCL-RS). The freeze-casting process was used to construct the porous PCL-RS/GAT scaffold after incorporating gelatin into the PCL-RS polymer solution. A scaffold with pores that are in perfect position and a porosity of 81.84% was successfully acquired. The biodegradability assessment, 48% of its early weight was lost after five weeks. The PCL-RS/GAT scaffold exhibited an elastic modulus of 32.01 MPa and a tensile strength of 3.9 MPa. The MTT analysis showed the PCL-RS/GAT favorable cytocompatibility with rat bone marrow-derived mesenchymal stem cells (BM-MSC). In addition, the cells cultured in the PCL-RS/GAT scaffold exhibited the greatest ALP activity and mineralization. The RT-PCR test results indicated that the PCL-RS/GAT scaffold had a high RUNX2, COL 1A1, and OCN gene expression, suggesting a strong osteoinductive capacity. The results indicate that the PCL-RS/GAT scaffold is a suitable biomimetic platform for tissue engineering in bone.

Synergistic integration of plant derived galactomannan and MXene to produce multifunctional nanocomposites with antibacterial and osteogenic properties

This study proposes to combine plant-derived galactomannan and MXene to form an organic-inorganic hydrogel network with integrative antibacterial and osteogenic properties. Oxidized galactomannan (OxGa) was blended with N-succinyl chitosan (NSC) and varying concentrations of Mxene, owing to its high reactivity and biocompatibility. The results indicated that Mxene-loaded OxGa/NSC scaffolds exhibited biomimetic topography, adequate mechanical features and excellent physicochemical properties with strong antibacterial properties against pathogenic microbes. The cell culture experiments showed dose-dependent cytotoxic behaviour. An optimized MXene content of ≤ 4 mg/ml revealed no cytotoxicity and augmented the proliferation of osteoblast cells. Similar results were obtained in gene expression analysis where ALP, COL1A1, RUNX2, and SOX2 genes showed up-regulation. In addition, the chorioallantoic membrane (CAM) assay also illustrated augmentation of angiogenesis without any toxicity. The overall results highlight the dual performance of OxGa/NSC-MXene scaffolds to exhibit anti-infection with simultaneous osteogenic properties, which is purely determined by the concentration of MXene. Therefore, regulating MXene concentration can serve as a chemical switch in imparting adequate antibacterial functions with the ability to allow new osseous generation. Our findings on the newly designed Mxene-loaded OxGa/NSC scaffolds can be a promising biomaterial for bone-related infections with a concurrent ability to promote osteogenesis.

Association of a COL1A1 Gene Haplotype with Pathologic Myopia in a Northern Chinese Han Population

To investigate the relationship between COL1A1 variations and the susceptibility to pathologic myopia (PM) among the general population in Northern China, we included 525 PM patients and 1105 non-myopic controls. All PM patients underwent comprehensive ophthalmologic examinations. DNA was extracted from peripheral venous blood samples and genotyped using the MassArray System. Statistical analyses, including Hardy-Weinberg equilibrium, χ2 test, and linkage disequilibrium analysis, were conducted to compare the genotypic and allelic distributions of SNPs between PM patients and controls. The results showed no significant differences in the genotypic and allelic distributions of rs2075555, rs2269336, and rs1107946 between the PM and control groups. However, haplotype analysis revealed that the G-G-C and T-C-A haplotypes are risk factors for PM (G-G-C: OR = 1.399, 95% CI = 1.206–1.623, P < 0.001, Pc < 0.001; T-C-A: OR = 1.248, 95% CI = 1.064–1.456, P = 0.007, Pc = 0.021). Although individual SNPs in COL1A1 were not significantly associated with PM, specific haplotypes (G-G-C and T-C-A) were identified as risk factors. This suggests a potential role of COL1A1 in the development of PM.

РАСПРЕДЕЛЕНИЕ ПОЛИМОРФНЫХ ВАРИАНТОВ ГЕНОВ ТРАНСФОРМИРУЮЩЕГО ФАКТОРА РОСТА Β1, Α1 ЦЕПИ КОЛЛАГЕНА IV ТИПА И ЭНДОТЕЛИНА-1 У ПАЦИЕНТОВ С АРТЕРИАЛЬНОЙ ГИПЕРТЕНЗИЕЙ

Background. Аn important role in the development and progression of arterial hypertension (AH) is played by the influence of polymorphic variants of genes. The polymorphisms C(-509)T, T869C and G915C of the transforming growth factor β1 (TGFB1) gene, A110186299G and C110196387T of the collagen type IV α1 chain (COL4A1) gene, G5665T of the endothelin-1 (EDN1) gene can contribute to the remodeling of the arterial wall. Objective: To assess the distribution of the polymorphic variants C(-509)T, T869C and G915C of the TGFB1 gene, A110186299G and C110196387T of the COL4A1 gene, G5665T of the EDN1 gene in male patients of 30-49 years old with hypertension living in the Grodno region. Material and methods: The study included 204 men (65 healthy individuals and 139 patients with grade 1 and 2 hypertension). Survey data (general clinical examinations, genotyping by polymerase chain reaction) were analyzed using the STATISTICA 10.0 program. Results. In patients with AH and in healthy men the frequency of occurrence of the T allele of the G5665T polymorphism was 16.8% and 13.9% (р=0.45), that of the T allele of the C(-509)T polymorphism – 34.2% and 36.9% (р=0.66) respectively. The frequency of the T allele of the T869C polymorphism was 41.4% and 40.8% (p=0.99), and that of the C allele of the G915C polymorphism – 7.9% and 6.9% (р=0.84) correspondingly. The frequency of the A allele of the polymorphism A110186299G comprised 35.6% and 37.7% (р=0.74), that of the T allele of the polymorphism C110196387T – 46.4% and 43.8% (р=0.63) respectively. Conclusions. The frequency of occurrence of genotypes and alleles of the studied polymorphisms is comparable among male patients with grade 1-2 AH and practically healthy individuals.

Tuning collective behaviour in zebrafish with genetic modification.

Zebrafish collective behaviour is widely used to assess their physical and mental state, serving as a valuable tool to assess the impact of ageing, disease genetics, and the effect of drugs. The essence of these macroscopic phenomena can be represented by active matter models, where the individuals are abstracted as interactive self-propelling agents. The behaviour of these agents depends on a set of parameters in a manner reminiscent of those between the constituents of physical systems. In a few cases, the system may be controlled at the level of the individual constituents such as the interactions between colloidal particles, or the enzymatic behaviour of de novo proteins. Usually, however, while the collective behaviour may be influenced by environmental factors, it typically cannot be changed at will. Here, we challenge this scenario in a biological context by genetically modifying zebrafish. We thus demonstrate the potential of genetic modification in the context of controlling the collective behaviour of biological active matter systems at the level of the constituents, rather than externally. In particular, we probe the effect of the lack of col11a2 gene in zebrafish, which causes the early onset of osteoarthritis. The resulting col11a2 -/- zebrafish exhibited compromised vertebral column properties, bent their body less while swimming, and took longer to change their orientations. Surprisingly, a group of 25 mutant fish exhibited more orderly collective motion than the wildtype. We show that the collective behaviour of wildtype and col11a2 -/- zebrafish are captured with a simple active matter model, in which the mutant fish are modelled by self-propelling agents with a higher orientational noise on average. In this way, we demonstrate the possibility of tuning a biological system, changing the state space it occupies when interpreted with a simple active matter model.

The BET protein inhibitor apabetalone (RVX-208) prevents doxorubicin-induced endothelial senescence

Abstract Background Accumulation of senescent endothelial cells (ECs) plays a pivotal role in cardiometabolic disorders and lifespan reduction(1). Yet, only a few senolytics are known to date, partly due to the poor grasp of the molecular mechanisms that control the senescence survival programme. Bromo-and extraterminal domain (BET) proteins, known epigenetic reader proteins, recognize acetylated histone tails and regulate gene transcription. RVX-208, an FDA-approved BET inhibitor, has shown to modulate transcriptional programs involved in inflammation, cancer, and renal disease(2). Yet, its potential role in EC senescence and cardio-oncology remains elusive. Purpose Our study aims to investigate whether pharmacological modulation of BET proteins by RVX-208 can mitigate doxorubicin-induced endothelial senescence. Methods Human aortic endothelial cells (HAECs) were exposed to different concentrations of Doxo (50nM-500nM) for 48 hours. Cell morphology changes, cell cycle arrest, senescence associated secreted phenotype (SASP) release, upregulation of SA-b gal activity, DNA damage response (gH2AX) and expression of p21, p16 and p53 were used as molecular markers of cellular senescence. Conditioned media was collected from Doxo-treated ECs and processed for molecular analyses or used for treatment of healthy, non-senescent ECs. To test whether RVX-208 exerts any senolytic or senomorphic effect, HAECs were treated with Doxo (100 nM) in presence or absence of RVX-208 (5mM-20mM) for 48 hrs. RNA-seq and proteomic profiling were performed in vehicle and Doxo-treated ECs. Results Doxo-treated ECs showed a dose-dependent increase in senescence markers (p16, p21 and p53) and DNA-damage response (gH2AX). FACS analysis revealed an accumulation of cells in G2/M phase in Doxo-treated ECs (100 nM). Of interest, treatment with RVX-208 (15mM) prevented the upregulation of senescent markers (p16, p21 and p53) while rescuing alterations in cell morphology and reducing the number of SA-b gal positive cells. RVX-208 prevented Doxo-induced upregulation of genes involved in inflammation, oxidative stress and mitochondrial dysfunction as shown by RNA-seq and proteomic analysis. Conditioned media from Doxo-treated ECs showed an enrichment of SASP-associated markers (IL6, CCL2, IL8, TIMP2 and PDGF-b) as shown by dot-blot arrays. Exposure of healthy ECs to conditioned media from senescent ECs recapitulated aging features. Notably, RVX-208 blunted the release of SASP markers (IL6, CCL2 and IL8) from Doxo-treated ECs. Mechanistically, we found enhanced BET protein occupancy (BRD4) with concomitant enrichment of activating chromatin marks (H3K27Ac and H3K4me) on the promoter of senescence genes COL1A2, CDKN1C and SESN3. Conclusion Targeting BET proteins with RVX-208 may offer a promising therapeutic strategy to prevent endothelial aging in cancer patients undergoing cardiotoxic therapies.

Dyspnea and nocturnal cough due to esophageal diffuse leiomyomatosis in a girl with hematuria.

A 7-year-old girl with hematuria and clinical suspicion of Alport syndrome (AS) presented with dyspnea and nocturnal cough, initially diagnosed and treated as asthma. Despite inhaled corticosteroid therapy, her symptoms persisted, and spirometry indicated obstructive lung function without bronchodilator response. Chest CT revealed diffuse thickening of the esophageal wall, tracheal compression, with involvement of the gastric cardia, suggestive of diffuse leiomyomatosis. Subsequent genetic reanalysis confirmed the presence of a contiguous deletion of COL4A5 and COL4A6 genes, solidifying the diagnosis of AS. Diffuse leiomyomatosis, a rare benign neoplasm associated with AS, typically manifests as dysphagia, but in this case, it presented initially with asthma-like symptoms. This case emphasizes the importance of imaging when asthma treatment fails, particularly in patients with coexisting conditions of another system.

Investigation of exon skipping therapy in kidney organoids from Alport syndrome patients derived iPSCs.

Alport syndrome (AS) is a hereditary disease caused by mutations in the COL4A5 gene and leads to chronic kidney disease. Currently, no specific treatment has been developed. However, a recent study using AS-model mice demonstrated that the exon skipping method could partially rescue the symptoms. In this study, we evaluated the effects of the exon skipping method using kidney organoids generated from AS-patient-derived induced pluripotent stem cells (AS-iPSCs). We generated kidney organoids from AS-iPSCs, which exhibited nephron structures. As expected, the C-terminus of COL4A5 was not expressed in AS-organoids. Interestingly, anti-sense oligonucleotides restored the expression of the C-terminus of COL4A5 in vitro. Next, we transplanted AS-organoids into mice and evaluated glomerular basement membrane formation in vivo. We found that AS-organoids formed a lower slit diaphragm ratio compared to control organoids. Finally, we assessed the effects of exon skipping on transplanted organoids but observed minimum effects. These studies suggest that AS-iPSCs can generate kidney organoids lacking the C-terminus of COL4A5, and that exon skipping can induce its expression in vitro.

Slowly progressive autosomal dominant Alport Syndrome due to COL4A3 splicing variant.

Alport syndrome is a rare genetic kidney disease caused by variants in the COL4A3/A4/A5 genes. It's characterised by progressive kidney failure, though therapies targeting Renin-Angiotensin System can delay its progression. Additionally, extrarenal manifestations may sometimes coexist. Recent advances in genetic analysis and the necessity to better clarify genotype-phenotype correlations in affected patients raises the importance of detecting even cryptic splicing variants, lying in both canonical and non-canonical splice sites variants such as last exonic nucleotide variants. These variants, often, do not cause an amino acid change but alter the snRNP proteins binding. We studied a big Italian family with Alport syndrome showing a clear dominant pattern of transmission with younger family members having only haematuria and older individuals presenting with End-Stage Kidney Failure (ESKF). Kidney biopsy showed the typical disease hallmarks. We deeply mined the data for SNV and CNV through exome sequencing on DNA from both peripheral blood samples and patients' podocytes-lineage cells. We identified an already reported synonymous variant, c.765G>A (p.(Thr255Thr)), in the last exonic nucleotide of exon 13 of the COL4A3 gene. Employing the patient's podocytes we demonstrated that this variant results in exon skipping leading to an in-frame deletion of 28 amino acids without leaky effect. According to the pattern of transmission, to the kidney biopsy and to the exome data analysis we provided further evidence that autosomal dominant Alport syndrome is a well-defined clinical entity. We also confirmed the pathogenicity of the synonymous COL4A3 variant for the first time demonstrating its role in a dominant pattern of transmission.

Effects of Cannabidiol on Intestinal Myofibroblast Fibrotic PathwaysPATHWAYS

Inflammatory bowel disease (IBD) includes two immune-mediated disorders, Crohn’s disease (CD) and ulcerative colitis (UC). IBD is characterized by recurrent and chronic inflammation of the gastrointestinal tract. IBD is lifelong, and there is no cure. There has been an interest in new advanced medications to decrease inflammation and manage symptoms. Many of these new medications are expensive and depress the immune system, leaving many patients looking for alternatives to current medical therapies. Some IBD patients use medical marijuana to relieve the symptoms of this disease, and cannabidiol (CBD) in particular shows potential for reducing inflammation. This study investigates the potential therapeutic effects of CBD on IBD by examining its impact on fibrotic pathways in human intestinal myofibroblast (InMyoFib) cells. Cultured InMyoFibs were pre-treated with CBD, followed by transforming growth factor-beta 1 (TGF-β1) to stimulate a pro-fibrotic phenotype and mimic human IBD in the lab. Expression of genes associated with fibrosis, inflammation, and cannabinoid signaling was measured by quantitative polymerase chain reaction (qPCR). Fibrotic protein expression in the culture media was analyzed using enzyme-linked immunosorbent assays (ELISA). CBD pre-treatment before TGF-β1 stimulation significantly reduced the expression of pro-fibrotic genes ACTA2, CCN2, COL1A1, and SERPINH1 compared to cells stimulated with TGF-β1 alone. Additionally, CBD increased the expression of inflammation-related genes IL6 and PTGS2 and the lipid metabolism gene PPARG, known to have anti-fibrotic properties. These findings suggest that CBD may modulate fibrotic and inflammatory signaling pathways, providing a potential therapeutic avenue for IBD treatment.

COL8A2 activation enhances function of corneal endothelial cells through HIPPO signaling/mitochondria pathway

Corneal endothelial cells (CECs) are essential for maintaining corneal transparency and hydration through their barrier and pump functions. The COL8A2 gene encodes a component of the extracellular matrix of the cornea, which is crucial for the normal functioning of these cells. Mutations in COL8A2 are linked to corneal dystrophies, emphasizing the gene's importance in corneal health. The purpose of this research is to explore the effects of COL8A2 activation within CECs, to understand its contribution to cellular behavior and health. COL8A2 CRISPR/dCas9 activation system (aCOL8A2) was used to activate the COL8A2. In rats, wound healing and mitochondrial function were assessed after COL8A2 activation. As a result, aCOL8A2 promoted wound healing of rat corneal endothelium by increasing mitochondrial membrane potential. In cultured human CECs, proteomic analysis was performed to screen and identify the differential protein profiles between control and aCOL8A2 cells. Western blot was used to validate the differential proteins from both cells. Mitochondrial function and intracellular distribution were assessed by measuring ATP production and mitochondrial membrane potential. In cultured human CECs, aCOL8A2 increased COL8A2 and phospho-YAP levels. Transendothelial electrical resistance (TEER) was increased and actin cytoskeleton was attenuated by aCOL8A2. Gene ontology analysis revealed that the proteins were mainly involved in the regulation of folate biosynthesis, ECM-receptor interaction, cell differentiation, NADP activity and cytoskeleton. ATP production was increased, mitochondrial membrane potential was polarized and mitochondrial distribution was widespread in the aCOL8A2 group. In conclusion, aCOL8A2 induces a regulatory cascade affecting mitochondrial positioning and efficiency, mediated by alterations in the cytoskeletal architecture and the YAP signaling pathway. This sequence of events serves to bolster the functional capacities of corneal endothelial cells, including their pump and barrier functions, essential for corneal health and transparency.

Identification of mutations using whole exome sequencing in eight fetuses presenting with short femur

BackgroundFemur length is one of the important indicators for evaluating fetal growth and development. Short femur is a common prenatal ultrasound finding. This study aimed to investigate the genetic etiology of fetal short femur using trio-based whole exome sequencing (WES), so as to provide evidence for prenatal diagnosis and evaluate the application of trio-WES in prenatal diagnosis of fetal short femur. MethodsWe retrospectively analyzed the clinical phenotype and WES results of eight fetuses with short femur diagnosed by prenatal ultrasound. The results of WES were validated by Sanger sequencing. The pathogenicity of the mutations was evaluated. Minigene assay was performed to investigate the effects of intronic mutation on mRNA splicing. The pregnancy outcome was followed up. ResultsA total of seven mutations were detected in eight short femur fetuses. Among them, COL2A1 (p.Gly1107Glu), GNAS (p.Lys739Glu) and FGFR3 (c.1075 + 95C > G) were novel mutations that had not been reported. Minigene assay showed that c.1075 + 95C > G in FGFR3 partially retained a 90 bp sequence in intron 8. ConclusionsThe results of this study enriched the mutant spectrums of COL2A1, GNAS and FGFR3 genes, and demonstrated the value of trio-WES in prenatal diagnosis of fetuses with short femur.