COL1A1 Gene Research Articles

Genetic and molecular dynamics analysis of two variants of the COL4A5 gene causing Alport syndrome

BackgroundAlport syndrome (AS; OMIM#308,940) is a hereditary kidney disease that progresses over time and is distinguished by hearing loss and ocular irregularities. The syndrome has three subtypes, namely X-linked (XL; OMIM#301,050), autosomal recessive (AR; OMIM#203,780), and autosomal dominant (AD; OMIM#104,200), which are categorized based on their respective modes of inheritance. XLAS is attributed to a pathogenic variant in the COL4A5 (OMIM*303,630) gene, which encodes the α5(IV) chain of type IV collagen (Col-IV). In contrast, ADAS and ARAS are the result of variants in the COL4A3 (OMIM*120,070) and COL4A4 (OMIM*120,131) genes, which encode the α3(IV) and α4(IV) chains of Col-IV, respectively. Typically, the diagnosis of AS necessitates hereditary or pathological assessments. The determination of splicing variants as pathogenic or non-pathogenic based on gene sequencing outcomes is challenging.MethodsIn this study, we conducted exome sequencing and Sanger sequencing on two unrelated Chinese patients with AS. We identified a deletion variant c.4414delG in the COL4A5 gene and a splicing variant c.4298-20T > A in the same gene. In order to ascertain the impact of c.4298-20T > A on the synthesis of COL4A5 mRNA, we performed experiments involving minigene splicing. Additionally, we predicted the ability of these two variants to affect triple helix formation of α345(IV) using molecular dynamics methods.ResultsThe c.4414delG deletion variant caused a change in the genetic code of the COL4A5 gene. Specifically, it caused a shift in codon 1472 from encoding aspartate to encoding methionine. This shift resulted in a change of 75 amino acids in the protein sequence, ultimately leading to an early stop codon. This premature stop codon caused the production of a truncated α5(IV) chain with a predicted protein effect of p.D1472Mfs. The mRNA of the COL4A5 gene experienced intron 46 retention due to the splicing variant c.4298-20T > A, leading to the inclusion of six additional amino acids between amino acids 1432 and 1433 of the α5(IV) chain. This variant is predicted to have a protein effect of p.(P1432_G1433insDYFVEI). The impact of two variants, c.4414delG and c.4298-20T > A, on the aggregation region for α3(IV), α4(IV), and α5(IV) trimerisation were studied using molecular dynamics simulations. Results showed that the deletion variant c.4414delG had a significantly stronger disruption on NC1, compared to the splicing variant c.4298-20T > A. This difference in impact is consistent with the varying clinical phenotypes observed in the two patients. Based on the American College of Medical Genetics and Genomics (ACMG) classification criteria and guidelines for genetic variants, the deletion variant c.4414delG was rated as pathogenic while the splicing variant c.4298-20T > A was rated as likely-pathogenic.ConclusionOur study has identified two novel pathogenic loci, the deletion variant c.4414delG and the splicing variant c.4298-20T > A, associated with XLAS. This finding expands the genetic spectrum of XLAS. We suggest that molecular dynamics can effectively model the effect of genetic variation on α345(IV) trimerization, which may offer valuable insights into the mechanisms of XLAS pathogenesis.

Exploration of Extracellular Vesicle Long Non-Coding RNAs in Serum of Patients with Cervical Cancer

Introduction: Cervical cancer (CC) is the fourth most common cancer type and a leading cause of cancer-related deaths in women worldwide. Its underlying molecular mechanisms are unclear. Cancer cell-derived extracellular vesicles (EVs) are involved in cancer development and progression by delivering regulatory factors, including microRNAs and long non-coding RNAs (lncRNAs). Methods: Here, we identified the EV lncRNA expression profiles associated with different developmental stages of CC using next-generation sequencing. EVs from the serum of patients with stages I–III CC and healthy donors were characterized using EV marker immunoblotting and transmission electron microscopy. Results: The EV concentration increases with progression of the disease. Most particles had a 100–250-nm diameter, and their sizes were similar in all groups. We identified many lncRNAs that were uniquely and differentially expressed (DE) in patients with different stages of CC. The pathway analysis results indicated that the upregulated DE EV lncRNAs abundant in stages I and II were associated with cell proliferation and inflammation and cancer progression pathways, respectively. LINC00941, LINC01910, LINC02454, and DSG2-AS1 were highly expressed, suggesting poor overall survival of CC patients. Interestingly, DSG2-AS1 was associated with the human papillomavirus infection pathway through AKT3, DLG1, and COL6A2 genes. Conclusion: This is the first study that reports the levels of EVs and their lncRNA contents change during cancer development, demonstrating the existence of a unique vesicle-mediated cell-to-cell communication network underlying cancer progression.

ЭФФЕКТИВНОСТЬ ТЕРАПИИ ИНГИБИТОРАМИ АНГИОТЕНЗИН- ПРЕВРАЩАЮЩЕГО ФЕРМЕНТА В ПРОФИЛАКТИКЕ РАЗВИТИЯ ПРОТЕИНУРИИ У МАЛЬЧИКОВ С Х-СЦЕПЛЕННЫМ СИНДРОМОМ АЛЬПОРТА: ДАННЫЕ ОДНОЦЕНТРОВОГО КОГОРТНОГО НЕРАНДОМИЗИРОВАННОГО ИССЛЕДОВАНИЯ

Patients with a hemizygous mutation in the COL4A5 gene have a high risk of developing renal failure at a young age. Proteinuria reflects the progression of glomerulopathy in Alport syndrome (AS). In order to improve renal survival in AS patients with proteinuria are prescribed with angiotensin system inhibitors. There is a limited, insufficient number of studies demonstrating the effectiveness of angiotensin-converting enzyme inhibitors (ACEI) in children with AS; the therapeutic effect initiated prior to the development of proteinuria has not been studied as yet. The purpose of the research was to determine the effectiveness of ACEI therapy in the form of enalapril in the prevention of proteinuria in boys with X-linked AS (XLAS). Materials and methods used: boys aged 2 to 15 y/o with genetically confirmed XLAS were included in a single-center prospective cohort study. Enalapril was prescribed at doses of 1 to 3 mg/m2/day. Urinary protein excretion (Pr) and estimated glomerular filtration rate (eGFR) were studied at the time of inclusion and with a frequency of one time per six months for the three-year period or longer. The primary endpoint was the time of onset of proteinuria. Results: 72 boys in total, incl. 29 without proteinuria, who received enalapril therapy (Group 1, age 6.2±1.9 y/o, Pr 67±20 mg/m2/day, eGFR 118±15.2 ml /min/1.73 m2) and 43 children without therapy (Group 2, age 6.8±1.9 y/o, Pr 87±23 mg/m2/day, eGFR 119±17.3 ml/min/1.73 m2). The incidence of proteinuria (0.45 in G1 vs. 0.98 in G2, p<0.001), incl. up to the age of 10 y/o (0.42 vs 0.91, respectively, p<0.001) was statistically significantly lower in G1. Proteinuria developed later in the treatment group (10.2±2.9 vs. 4.9±1.4 years, p=0.300). Non-missense mutations in the COL4A5 gene, episodes of macrohematuria in records and lack of therapy were the risk factors for proteinuria; the lack of enalapril therapy was a predictor for proteinuria. The risk of proteinuria during therapy was reduced by 50% (RR=0.46 (95% CI 0.31; 0.68); ARR=0.53; NNT=1.9). Conclusion: initiation of enalapril therapy at the pre-proteinuria stage of nephropathy in boys with XLAS reduces the risk of proteinuria preventing its occurrence in every 50$ of cases when receiving treatment. Early initiation of therapy may be effective in preventing the progression of kidney disease in boys with XLAS.

The Action of Angiocrine Molecules Sourced from Mechanotransduction-Related Endothelial Cell Partially Explain the Successful of Titanium in Osseointegration.

Since Branemark's findings, titanium-based alloys have been widely used in implantology. However, their success in dental implants is not known when considering the heterogenicity of housing cells surrounding the peri-implant microenvironment. Additionally, they are expected to recapitulate the physiological coupling between endothelial cells and osteoblasts during appositional bone growth during osseointegration. To investigate whether this crosstalk was happening in this context, we considered the mechanotransduction-related endothelial cell signaling underlying laminar shear stress (up to 3 days), and this angiocrine factor-enriched medium was harvested further to use exposing pre-osteoblasts (pOb) for up to 7 days in vitro. Two titanium surfaces were considered, as follows: double acid etching treatment (w_DAE) and machined surfaces (wo_DAE). These surfaces were used to conditionate the cell culture medium as recommended by ISO10993-5:2016, and this titanium-enriched medium was later used to expose ECs. First, our data showed that there is a difference between the surfaces in releasing Ti molecules to the medium, providing very dynamic surfaces, where the w_DAE was around 25% higher (4 ng/mL) in comparison to the wo_DAE (3 ng/mL). Importantly, the ECs took up some of this titanium content for up to 3 days in culture. However, when this conditioned medium was used to expose pOb for up to 7 days, considering the angiocrine factors released from ECs, the concentration of Ti was lesser than previously reported, reaching around 1 ng/mL and 2 ng/mL, respectively. Thereafter, pOb exposed to this angiocrine factor-enriched medium presented a significant difference when considering the mechanosignaling subjected to the ECs. Shear-stressed ECs showed adequate crosstalk with osteoblasts, stimulating the higher expression of the Runx2 gene and driving higher expressions of Alkaline phosphatase (ALP), bone sialoprotein (BSP), and osteocalcin. Mechanotransduction-related endothelial cell signaling as a source of angiocrine molecules also stimulated the higher expression of the Col3A1 gene in osteoblasts, which suggests it is a relevant protagonist during trabecular bone growth. In fact, we investigated ECM remodeling by first evaluating the expression of genes related to it, and our data showed a higher expression of matrix metalloproteinase (MMP) 2 and MMP9 in response to mechanosignaling-based angiocrine molecules, independent of considering w_DAE or the wo_DAE, and this profile reflected on the MMP2 and MMP9 activities evaluated via gelatin-based zymography. Complimentarily, the ECM remodeling seemed to be a very regulated mechanism in mature osteoblasts during the mineralization process once both TIMP metallopeptidase inhibitor 1 and 2 (TIMP1 and TIMP2, respectively) genes were significantly higher in response to mechanotransduction-related endothelial cell signaling as a source of angiocrine molecules. Altogether, our data show the relevance of mechanosignaling in favoring ECs' release of bioactive factors peri-implant, which is responsible for creating an osteogenic microenvironment able to drive osteoblast differentiation and modulate ECM remodeling. Taking this into account, it seems that mechanotransduction-based angiocrine molecules explain the successful use of titanium during osseointegration.

Chronic exposure to environmentally relevant concentration of fluoride impairs osteoblast's collagen synthesis and matrix mineralization: Involvement of epigenetic regulation in skeletal fluorosis

Globally, 200 million people are suffering from toxic manifestations of Fluoride(F), dental and skeletal fluorosis; unfortunately, there is no treatment. To unravel the pathogenesis of skeletal fluorosis, we established fluorosis mice by treating environmentally relevant concentration of F (15 ppm NaF) through drinking water for 4 months. As in skeletal fluorosis, locomotor disability, crippling deformities occur and thus, our hypothesis was F might adversely affects collagen which gives the bone tensile strength. This work inevitably had to be carried out on osteoblast cells, responsible for synthesis, deposition, and mineralization of bone matrix. Isolated osteoblast cells were confirmed by ALP activity and mineralized nodules formation. Expression of collagen Col1a1, Col1a2, COL1A1 was significantly reduced in treated mice. Further, a study revealed the involvement of epigenetic regulation by promoter hypermethylation of Col1a1; expressional alterations of transcription factors, calcium channels and other genes e.g., Cbfa-1, Tgf-β1, Bmp1, Sp1, Sp7, Nf-Kb p65, Bmp-2, Bglap, Gprc6a and Cav1.2 are associated with impairment of collagen synthesis, deposition and decreased mineralization thus, enfeebling bone health. This study indicates the possible association of epigenetic regulation in skeletal fluorosis. However, no association was found between polymorphisms in the Col1a1 (RsaI, HindIII) and Col1a2 (RsaI, HindIII) genes with fluorosis in mice.

Abstract P3049: Exploring The Therapeutic Potential Of Aav-mediated Restoration Of Col6a2 In A Mouse Model Of Ullrich Muscular Dystrophy

Ullrich muscular dystrophy is a genetic disease characterized by joint contractures and muscle weakness which leads to loss of ambulation and respiratory insufficiency early in life. This disease is caused by mutations in any of the three major genes of collagen VI - Col6a1, Col6a2, or Col6a3. Together, these three genes are required to generate a complex quaternary protein assembly that is secreted to help form the basement membrane. Currently there is no cure or greater treatment for Ullrich muscular dystrophy although 2 of the genes (Col6a1, Col6a2) are within the size limit of adeno-associated virus (AAV) and could be addressed by gene therapy. However, it is unclear what cell type in muscle generates the bulk of Collagen VI during development, or if it could be replaced at a later time by gene therapy from expression in myofibers or from fibroblasts. Given that mutations in the Col6a2 gene are the most common cause of Ullrich muscular dystrophy, here we generated Col6a2 gene deleted (Col6a2 -/- ) mice which showed the anticipated histopathologic evidence of dystrophic disease as young adults. Adult mice were then subject to retro-orbital injection of MyoAAV-Col6a2, which resulted in robust expression of Col6a2 within the myofibers of all skeletal muscles analyzed (and heart), although this was ineffective in restoring the collagen VI complex within the basement membrane. Further characterization into the effects of Col6a2 re-expression from the myofibers versus the fibroblasts in these gene targeted mice is underway with the goal of defining a combinatorial (with Col6a1 and Col6a3) or cell type restricted infection protocol to successfully achieve full restoration of Collagen VI in muscle.

Genotypic Analysis of COL4A1 Gene in Diabetic Nephropathy and Type 2 Diabetes Mellitus Patients: A Comparative Genetic Study.

Diabetic nephropathy (DN) is specified by microalbuminuria, glomerular lesions, and renal fibrosis leading to end-stage renal disease. The pathophysiology of DN is multifactorial as a result of gene-environment interaction. Clinical studies suggested that gene mutations affect various pathways involved in DN, including extracellular matrix (ECM). During chronic hyperglycemia, collagen type-4-mediated ECM overproduction occurs, leading to renal fibrosis and DN development. In this study, COL4A1 gene variant rs605143 (G/A) was analyzed in diabetes and DN patients from the study population. We genotyped 386 study subjects, comprising 120 type 2 diabetes mellitus (T2DM) patients, 120 DN, and 146 healthy controls. All study subjects were analyzed for biochemical assays by commercially available kits and genotypic analysis by polymerase chain reaction-restriction fragment length polymorphism and confirmed by Sanger sequencing. Statistical analyses were done using SPSS and GraphPad. Anthroclinicopathological parameters showed a significant association between T2DM and DN. Genotype AA of COL4A1 gene variant rs605143 (G/A) showed a significant association with T2DM and DN compared with controls with 5.87- and 8.01-folds risk, respectively. Mutant allele A also significantly associated with T2DM and DN independently compared with healthy controls with 2.29- and 2.81-time risk in the study population. This study's findings suggested that COL4A1 gene variant rs605143 (G/A) can be used as predictive biomarkers for T2DM and DN independently. However, this gene variant needs to be analyzed in a large sample to explore the shared genetic association between T2DM and DN.

Liver fibrotic development is reduced through inflammation prevention by an adenosine derivative compound

BackgroundLiver fibrosis is a global health problem, and studying its development provides important information to address its treatment. Here, we characterized the effects of an adenosine compound (IFC-305) on preventing fibrosis and liver inflammation. Methods: We studied the impact of IFC-305 on a carbon tetrachloride-induced liver fibrosis model in Wistar male rats at 4, 6, and 8 weeks. The effects were characterized by liver tissue histology, macrophages identification by flow cytometry with CD163+/CD11b/c+ antibodies, hepatic and plasmatic cytokine levels employing MILLIPLEX MAP and ELISA, Col1a1 and Il6 gene expression by RTqPCR, lipoperoxidation by TBARS reaction, and reactive oxygen species using 2'−7'dichlorofluorescin diacetate. Results: CCl4-induced liver fibrosis and inflammation were significantly reduced in rats treated with IFC-305 at 6 and 8 weeks. In addition, we observed diminished expression of Col1a1; a decrease in the inflammatory cytokines IL-1β, IL-6, MCP-1, TNF-α, and IL-4 a; reduction in inflammatory macrophages; inhibition of lipoperoxidation; and ROS production in Kupffer cells. Conclusion: This study showed that IFC-305 can inhibit liver fibrosis establishment by regulating the immune response during CCl4-induced damage. The immunomodulatory action of IFC-305 supports its use as a potential therapeutic strategy for preventing liver fibrosis.

Patient-derived extracellular matrix demonstrates role of COL3A1 in blood vessel mechanics.

Vascular Ehlers-Danlos Syndrome (vEDS) is a rare autosomal dominant disease caused by mutations in the COL3A1 gene, which renders patients susceptible to aneurysm and arterial dissection and rupture. To determine the role of COL3A1 variants in the biochemical and biophysical properties of human arterial ECM, we developed a method for synthesizing ECM directly from vEDS donor fibroblasts. We found that the protein content of the ECM generated from vEDS donor fibroblasts differed significantly from ECM from healthy donors, including upregulation of collagen subtypes and other proteins related to ECM structural integrity. We further found that ECM generated from a donor with a glycine substitution mutation was characterized by increased glycosaminoglycan content and unique viscoelastic mechanical properties, including increased time constant for stress relaxation, resulting in a decrease in migratory speed of human aortic endothelial cells when seeded on the ECM. Collectively, these results demonstrate that vEDS patient-derived fibroblasts harboring COL3A1 mutations synthesize ECM that differs in composition, structure, and mechanical properties from healthy donors. These results further suggest that ECM mechanical properties could serve as a prognostic indicator for patients with vEDS, and the insights provided by the approach demonstrate the broader utility of cell-derived ECM in disease modeling. STATEMENT OF SIGNIFICANCE: The role of collagen III ECM mechanics remains unclear, despite reported roles in diseases including fibrosis and cancer. Here, we generate fibrous, collagen-rich ECM from primary donor cells from patients with vascular Ehlers-Danlos syndrome (vEDS), a disease caused by mutations in the gene that encodes collagen III. We observe that ECM grown from vEDS patients is characterized by unique mechanical signatures, including altered viscoelastic properties. By quantifying the structural, biochemical, and mechanical properties of patient-derived ECM, we identify potential drug targets for vEDS, while defining a role for collagen III in ECM mechanics more broadly. Furthermore, the structure/function relationships of collagen III in ECM assembly and mechanics will inform the design of substrates for tissue engineering and regenerative medicine.

Evaluation of COL1A1 gene rs1800012 polymorphism and non-differentiated connective tissue dysplasia as predictors of pelvic organ prolapse

The article presents data on the association of the rs1800012 polymorphism of the procollagen type I alpha (α) 1 COL1A1 gene and the stigma of non-differentiated connective tissue dysplasia with the development of vaginal prolapse in the Ukrainian population.The objective: to determine the prognostic significance of the rs1800012 polymorphism of the procollagen type I alpha (α) 1 COL1A1 gene and the clinical and anamnestic stigmata of non-differentiated connective tissue dysplasia as factors associated with the development of vaginal prolapse in the Ukrainian population.Materials and methods. In order to achieve the research aim, polymorphism in the rs1800012 locus of the COL1A1 gene was studied by polymerase chain reaction in venous blood samples of 120 women. The main group included 60 patients with vaginal prolapse III, IV, who had indications for surgical correction; to the comparison group – 60 women without pelvic floor dysfunction.The diagnostic algorithm included a physical examination, taking the general and gynecological history, evaluation of the state of the pelvic floor and urogenital dysfunction. The severity of pelvic floor prolapse was assessed based on the Pelvic Organ Prolapse Quantification (POP-Q) classification.Results. During the analysis of clinical and anamnestic data in women of both research groups, concomitant pathology with a possible connection with signs of non-differentiated connective tissue dysplasia was identified as risk factors for the development of pelvic floor dysfunction: 37 people in the main group versus 21 patients in the comparison group (χ2=8.5428; p=.003469).The prevalence of rs1800012 polymorphism of the gene procollagen type I alpha (α) 1 COL1A1 in the homozygous variant (TT) in this study was found in the main group in 8 samples (13.33%) and only in 2 (3.33%) – in the comparison group. The presented polymorphism in the heterozygous variant (GT) in the main group was determined in 19 samples (31.67%) and only in 12 (20.00%) – in the comparison group (χ2=7.3199; p=.025734).Conclusions. The association of the rs1800012 polymorphism in the COL1A1 gene (p=.025734) and clinical and anamnestic stigmata of non-differentiated connective tissue dysplasia (NDST) (p=.003469) with the development of pelvic organ prolapse was established, which is evidence of the importance of genetic changes and connective tissue remodeling in the etiology of genital prolapseThe presence of NDST stigma increases the risk of pelvic organ prolapse by 3 times (OR=3.788; 95% CI: 1.664–8.624), polymorphism rs1800012 in the COL1A1 gene – by 2 times with the heterozygous variant (OR=2.400; 95% CI: 1.230–6.633) and 3 times with the homozygous variant (OR=3.275; 95% CI: 1.300–19.969).

Extracellular matrix of ameloblastoma-derived negatively regulates osteogenic differentiation.

To investigate the effects of key pathogenic genes involved in the development of jaw ameloblastoma (AB) and its associated extracellular matrix (ECM) on osteogenic differentiation in order to provide a theoretical foundation for future research into bone aggressiveness of AB. The essential genes were identified by five AB patients for whole-exome sequencing and the microarray datasets GES38494 and GES132472. Moreover, the expression of key genes and their encoded proteins in AB tissues was explored. In addition, AB-derived the decellularized ECM (ABdECM) tissues were generated by the decellularization technique. Furthermore, the osteogenic development of periodontal ligament stem cells (PDLSCs) was mimicked by simulating the effects of the AB tumor microenvironment (TME). The AB essential genes including COL1A2, COL4A2, FBN1, and HPSE were discovered. Among them, the expression of HPSEwas down-regulated, while that of COL1A2, COL4A2, and FBN1 was noticeably upregulated in AB compared with normal gingival tissues of the jaws. In vitro osteogenic differentiation of PDLSCs was suppressed by the ABdECM. Abnormal ECM proteins encoded by COL4A2, COL1A2, FBN1, and HPSE genes can cause disturbance in the ECM environment of AB and promote bone resorption.

Chronic-binge ethanol feeding aggravates systemic dyslipidemia in Ldlr-/- mice, thereby accelerating hepatic fibrosis.

Chronic ethanol consumption is known to cause alcohol-associated liver disease, which poses a global health concern as almost a quarter of heavy drinkers develop severe liver damage. Alcohol-induced liver disease ranges from a mild, reversible steatotic liver to alcoholic steatohepatitis and irreversible liver fibrosis and cirrhosis, ultimately requiring liver transplantation. While ethanol consumption is associated with dysregulated lipid metabolism and altered cholesterol homeostasis, the impact of dyslipidemia and pre-existing hypercholesterolemia on the development of alcohol-associated liver disease remains to be elucidated. To address the influence of systemic dyslipidemia on ethanol-induced liver disease, chronic-binge ethanol feeding was applied to female C57BL/6J (wild type) mice and mice deficient for the low-density lipoprotein receptor (Ldlr-/-), which display a human-like lipoprotein profile with elevated cholesterol and triglyceride levels in circulation. Respective control groups were pair-fed an isocaloric diet. Chronic-binge ethanol feeding did not alter systemic lipid levels in wild type mice. While increased systemic cholesterol levels in Ldlr-/- mice were not affected by ethanol feeding, chronic-binge ethanol diet aggravated elevated plasma triglyceride levels in Ldlr-/- mice. Despite higher circulatory triglyceride levels in Ldlr-/- mice, hepatic lipid levels and the development of hepatic steatosis were not different from wild type mice after ethanol diet, while hepatic expression of genes related to lipid metabolism (Lpl) and transport (Cd36) showed minor changes. Immunohistochemical assessment indicated a lower induction of infiltrating neutrophils in the livers of ethanol-fed Ldlr-/- mice compared to wild type mice. In line, hepatic mRNA levels of the pro-inflammatory genes Ly6g, Cd11b, Ccr2, Cxcl1 and F4/80 were reduced, indicating less inflammation in the livers of Ldlr-/- mice which was associated with reduced Tlr9 induction. While systemic ALT and hepatic MDA levels were not different, Ldlr-deficient mice showed accelerated liver fibrosis development after chronic-binge ethanol diet than wild type mice, as indicated by increased levels of Sirius Red staining and higher expression of pro-fibrotic genes Tgfb, Col1a1 and Col3a1. Ldlr-/- and wild type mice had similar plasma ethanol levels and did not show differences in the hepatic mRNA levels of Adh1 and Cyp2e1, important for ethanol metabolism. Our results highlight that chronic-binge ethanol feeding enhances systemic dyslipidemia in Ldlr-/- mice which might accelerate the development of hepatic fibrosis, independent of hepatic lipid levels.

Biopolymer collagen-chitosan scaffold containing Aloe vera for chondrogenic efficacy on cartilage tissue engineering

The chondrogenic efficacy of aloe vera blended collagen-chitosan (COL-CS-AV) porous scaffold was investigated using articular chondrocytes in a standard condition. Cytocompatibility was analyzed using fluorescent dyes (calcein AM/ethidium bromide) and the viable cells were quantified by MTT assay. Glycosaminoglycan (GAG) content of ECM was estimated by using 1, 9-Dimethyl methylene Blue (DMMB). The total RNA content was quantified and the cartilage specific genes (col2a1, Acan) were amplified by reverse transcription-PCR from the cell lysate of the scaffolds. Histological examination was made using Haematoxylin and Eosin (H&E), safranin-O, masson's trichrome, alcian blue, and alizarin red to stain the specific component of ECM secreted on the construct. The cartilage specific collagen type II was estimated by immunohistochemistry using monoclonal type II collagen antibody. The results of these studies proved that COL-CS-AV scaffold has more chondrogenic efficacy than COL-CS, thus the aloe vera blend COL-CS-AV scaffold might be used as suitable candidate for cartilage tissue engineering.

Czech dysplasia mimicking rheumatoid arthritis: Case series andliterature review.

This study reported a family with most members affected by Czech dysplasia. We examined the patients' clinical, laboratory, and imaging characteristics and evaluated their functional capacity using the Stanford Health Assessment Questionnaire-Disability Index. The method used was case series description and literature review. This study showed that the pathogenic variant c.823C>T in the COL2A1 gene, which is a characteristic of Czech dysplasia, was found in 12 Brazilian individuals. Half of the patients in this family met the criteria for rheumatoid arthritis (RA) based on the 2010 American College of Rheumatology/European League Against Rheumatism classification criteria. Patients had arthritis in their hand joints, synovitis detected by ultrasound, and alterations in inflammatory tests. The Stanford Health Assessment Questionnaire-Disability Index assessment revealed that all patients exhibited moderate-to-severe functional disability. What distinguish Czech dysplasia from RA are an autosomal dominant inheritance pattern, platyspondyly, sensorineural hearing loss, and shortening of the metatarsal bones. It is important to consider Czech dysplasia as a potential differential diagnosis for RA. This autosomal dominant skeletal dysplasia is associated with normal height, short metatarsals, platyspondyly, hearing loss, enlarged epiphyses, and precocious osteoarthritis. Inflammatory findings such as arthritis, synovitis, and alteration of inflammatory markers may also be present in individuals with Czech dysplasia.

Whole exome sequencing identifies a novel variant in the COL12A1 gene in a family with Ullrich congenital muscular dystrophy 2.

Mutations within the COL12A1 gene have been linked with the onset of congenital Ullrich muscular dystrophy 2 (UCMD2) and Bethlem myopathy. The severity of the symptoms exhibited is dependent on the mutation's type and whether it is heterozygous or homozygous. We used whole-exome sequencing to identify disease-causing variants in a nine-year-old Iranian patient who had weakness, joint contractures, delayed motor development, and other symptoms. We confirmed the pathogenicity of the identified variant using in silico tools and verified its novelty using various databases. We also performed a co-segregation study and confirmed the presence of the variant in the patient's parents by Sanger sequencing. Our analysis identified a novel homozygous missense variant in the affected patient in COL12A1 (c.8828C > T; p.Pro2943Leu). This is the second reported family with UCMD2 caused by a mutation in COL12A1. Our findings confirm that this mutation results in significantly more severe symptoms than Bethlem myopathy. Our investigation contributes to the expanding body of evidence that links mutations in COL12A1 with UCMD2. Our findings confirm that the homozygous mutation in COL12A1 caused this condition and suggest that genetic testing for this mutation may be useful for diagnosing patients with this disease.

A Novel Cell Membrane-Associated RNA Extraction Method and Its Application in the Discovery of Breast Cancer Markers.

Cell membrane-associated RNA (mem-RNA) has been demonstrated to be cell-specific and disease-related and are considered as potential biomarkers for disease diagnostics, drug delivery, and cell screening. However, there is still a lack of methods specifically designed to extract mem-RNA from cells, limiting the discovery and applications of mem-RNA. In this study, we propose the first all-in-one solution for high-purity mem-RNA isolation based on two types of magnetic nanoparticles, named MREMB (Membrane-associated RNA Extraction based on Magnetic Beads), which achieved ten times enrichment of cell membrane components and over 90% recovery rate of RNA extraction. To demonstrate MREMB's potential in clinical research, we extracted and sequenced mem-RNA of typical breast cancer MCF-7, MDA-MB-231, and SKBR-3 cell lines and non-neoplastic breast epithelial cell MCF-10A. Compared to total RNA, sequencing results revealed that membrane/secreted protein-encoding mRNAs and long noncoding RNAs (lncRNAs) were enriched in the mem-RNA, some of which were significantly overexpressed in the three cancer cell lines, including extracellular matrix-related genes COL5A1 and lncRNA TALAM1. The results indicated that MREMB could enrich membrane/secreted protein-coding RNA and amplify the expression differences of related RNAs between cancer and non-neoplastic cells, promising for cancer biomarker discovery.

COL4A5 genotypes and clinical characteristics of children with Alport syndrome

To investigate the genotypes of the pathogenic gene COL4A5 and the characteristics of clinical phenotypes in children with Alport syndrome (AS). A retrospective analysis was performed for the genetic testing results and clinical data of 19 AS children with COL4A5 gene mutations. Among the 19 children with AS caused by COL4A5 gene mutations, 1 (5%) carried a new mutation of the COL4A5 gene, i.e., c.3372A>G(p.P1124=) and presented with AS coexisting with IgA vasculitis nephritis; 3 children (16%) had large fragment deletion of the COL4A5 gene, among whom 2 children (case 7 had a new mutation site of loss51-53) had gross hematuria and albuminuria at the onset, and 1 child (case 13 had a new mutation site of loss3-53) only had microscopic hematuria, while the other 15 children (79%) had common clinical phenotypes of AS, among whom 7 carried new mutations of the COL4A5 gene. Among all 19 children, 3 children (16%) who carried COL4A5 gene mutations also had COL4A4 gene mutations, and 1 child (5%) had COL4A3 gene mutations. Among these children with double gene mutations, 2 had gross hematuria and proteinuria at the onset. This study expands the genotype and phenotype spectrums of the pathogenic gene COL4A5 for AS. Children with large fragment deletion of the COL4A5 gene or double gene mutations of COL4A5 with COL4A3 or COL4A4 tend to have more serious clinical manifestations.

Phenotype and genotype correlation of inherited epidermolysis bullosa in Indonesia.

Inherited epidermolysis bullosa (EB) is a group of genodermatoses with considerable clinical and genetic heterogeneity. Clinical diagnosis of the EB subtypes is frequently imprecise and requires confirmation with genetic testing. There is still limited study using genetic testing to identify EB subtypes in Indonesia. This study aims to identify the pathogenic variants of inherited EB patients at the Department of Dermatology and Venereology, Universitas Padjadjaran-Dr Hasan Sadikin General Hospital in Bandung, West Java, Indonesia and to describe the correlation between the phenotype and genotype of our patients. Twelve patients clinically diagnosed with EB were included in this study. Genetic testing was performed in collaboration with KK Women's and Children's Hospital, Singapore. Pathogenic variants were identified in the COL7A1 gene in seven patients, namely Dominant Dystrophic EB (DDEB) with mutation types c.5945G>T, c.6218G>A, Recessive Dystrophic EB (RDEB) c.2005C>T, c.6081dup, c.1268C>T, c.1784C>T which are all known mutations. Novel mutations were found in the COL7A1 gene in two patients namely DDEB c.6253G>T and RDEB c.6740C>T. Two EB Simplex (EBS) patients showed mutation KRT14 gene as c.356T>C, c.373C>T which are known mutation. In addition, a novel mutation in LAMA3 gene c.2649del was found in one Junctional EB (JEB) patient. The molecular diagnoses of 12 Indonesian EB patients were identified, of which three were novel pathogenic variants. Concordance between the initial clinical diagnosis and genetic testing was only 33%. This demonstrated the importance of early genetic testing for accurate diagnosis, prognostication, management and genetic counselling.

Investigation of a family affected by early-onset osteoarthritis – proposal of a clinical pathway and bioinformatics pipeline for the investigation of cases of familial OA

BackgroundFamilial cases of early-onset osteoarthritis (OA) are rare although the exact prevalence is unknown. Early recognition of underlying OA-associated disorders is vital for targeted treatment, when available, and genetic counselling, in case of skeletal dysplasias. Currently, there is no clear guidance on how best to investigate families affected by early-onset OA.MethodsWe investigated a family with multiple members affected by early-onset OA (age at onset ≤ 40 years). Clinical and demographic characteristics were collected, followed by laboratory investigations screening for a range of potential OA-associated disorders, and whole genome sequencing in selected individuals.ResultsSeventeen members of the family were included (7 affected and 10 non-affected). There was an even split between the two sexes and two participants were under 18 years old. No pattern of abnormality was seen in the laboratory investigation that could explain the OA phenotype in the family. Whole-genome sequencing was perfomed in one participant and analysed for likely pathogenic variants in genes known to be associated with skeletal dysplasias. A heterozygous variant in the COL2A1 gene was identified (p.Arg519Cys). Confirmatory tests were performed in five additional participants (four affected and one unaffected).ConclusionThe methodology used in this study, including the clinical pathway and bioinformatics pipeline, could be applied to other families affected by early-onset OA.

Analysis of COL1A1 and COL1A2 gene variants in two fetuses with osteogenesis imperfecta

To explore the genetic basis of two fetuses with an osteogenesis imperfecta (OI) phenotype. Two fetuses diagnosed at the Affiliated Hospital of Weifang Medical College respectively on June 11, 2021 and October 16, 2021 were selected as the study subjects. Clinical data of the fetuses were collected. Amniotic fluid samples of the fetuses and peripheral blood samples of their pedigree members were collected for the extraction of genomic DNA. Whole exome sequencing (WES) and Sanger sequencing were carried out to identify the candidate variants. Minigene splicing reporter analysis was used to validate the variant which may affect the pre-mRNA splicing. For fetus 1, ultrasonography at 17+6 weeks of gestation had revealed shortening of bilateral humerus and femurs by more than two weeks, in addition with multiple fractures and angular deformities of long bones. WES revealed that fetus 1 had harbored a heterozygous c.3949_3950insGGCATGT (p.N1317Rfs*114) variant in exon 49 of the COL1A1 gene (NM_000088.4). Based on the guidelines from the American College of Medical Genetics and Genomics (ACMG), it was classified as a pathogenic variant (PVS1+PS2+PM2_Supporting) for disrupting the downstream open reading frame resulting in premature translational termination, being de novo in origin, and lacking records in the population and disease databases.For fetus 2, ultrasonography at 23 weeks of gestation also revealed shortening of bilateral humerus and femurs by one and four weeks, respectively, in addition with bending of bilateral femurs, tibias and fibulas. Fetus 2 had harbored a heterozygous c.1557+3A>G variant in intron 26 of the COL1A2 gene (NM_000089.4). Minigene experiment showed that it has induced skipping of exon 26 from the COL1A2 mRNA transcript, resulting in an in-frame deletion (c.1504_1557del) of the COL1A2 mRNA transcript. The variant was inherited from its father and had been previously reported in a family with OI type 4. It was therefore classified as a pathogenic variant (PS3+PM1+PM2_Supporting+PP3+PP5). The c.3949_3950insGGCATGT (p.N1317Rfs*114) variant in the COL1A1 gene and c.1557+3A>G variant in the COL1A2 gene probably underlay the disease in the two fetuses. Above findings not only have enriched the mutational spectrum of OI, but also shed light on the correlation between its genotype and phenotype and provided a basis for genetic counseling and prenatal diagnosis for the affected pedigrees.