COL1A1 Gene Research Articles

#1468 Alport syndrome family screening and management—experience of a tertiary center

Abstract Background and Aims Alport syndrome (AS) is the most common cause of inherited chronic kidney disease. This disorder is caused by deleterious variants on COL4A3, COL4A4 or COL4A5 genes. These genes codify the proteins that constitute collagen type IV of the glomerular basement membrane (GBM). Importantly, AS patients can develop chronic kidney disease (CKD) kidney disease that can be delayed or possibly prevented by timely initiation of therapy. The identification of at-risk relatives and their genetic testing allow to identify AS patients and initiate appropriate renal care and avoid unnecessary vigilance if the test is negative. Our aim was to assess the outcomes of Alport Syndrome cascade family screening in a tertiary hospital. Method In this study, we retrospectively assessed familial relatives at risk of AS index cases in our Nephrogenetics consult, from January 2018 to November 2023. After referral, routine tests to detect renal disease were done and AS gene panel was performed. Demographic and clinical data were also compiled. Results A total of 75 at-risk relatives were assessed, belonging to 26 families with a COL4A variant identified in the index case. Relatives at risk were mostly referred by primary care doctors (n = 52, 69.3%), with other 15 patients (20%) directly scheduled for consultation, and 8 (10.7%) referred by a nephrology clinic. In this cohort, an AS diagnosis was confirmed in 43 relatives (57.3%), with an additional 13 (17.3%) of the at-risk relatives with ongoing biochemical or molecular analyses. In 19 (25.4%) relatives the molecular study allowed the exclusion of AS. In the subgroup of patients diagnosed with AS (n = 43): median age at molecular diagnosis was 40.5 years (IQR 20.2-56.5years) and 26 (60.5%) were female. Thirty-five patients (81.4%) patients had autosomal dominant AS: 32 had a variant in COL4A3 (6 different variants were found: 4 were pathogenic or likely pathogenic and 2 were of uncertain significance) and 3 patients on COL4A4. COL4A5 variants were identified in 8 patients, 5 of them were female. Three (7%) patients presented with intermittent hematuria, 14 (32.6%) patients presented isolated hematuria, 10 (23.2%) patients had urinary protein/creatine (Up/cr) <0,3g/g, 7 (16.2%) presented Up/cr between 0.3g/g and 1g/g, 3 (7%) patients had Up/cr >1g/g; Six (14%) had CKD. Twenty-two (51.2%) patients were started on a renin angiotensin system inhibitor (RASi) and 5 (11.6%) were already under a RASi. At the last visit in this group of AS patients, the mean epiTFG was 104.2 ml/min (IQ 85.6-120-5 ml/min) and 6 (14%%) patients had epiTFG below 60 ml/min; mean Uprot/creat was 0.11g/g (IQR 0.06-0.32), 12 (28%) patients had no proteinuria. In the subgroup of relatives at risk were AS excluded (n = 19): median age was 38.5 years (IQR 20.2-56.0) and 11 (58%) were female. One patient had CKD, 7 (36.8%) had no signs of renal disease and 8 (42%) patients had intermittent hematuria. Conclusion Our retrospective review showed that the systematic screening of relatives at risk in our hospital was met with a high discovery rate of AS patients. Since there is clinical benefit in an early AS diagnosis, namely through improved renal care and prevention of disease progression, timely genetic/biochemical screening of at-risk relatives is advisable. Additionally, by discharging the relatives with a negative test, this is a more cost-effective approach than providing continuous vigilance to all.

#1609 Effect of dapagliflozin in proteinuria reduction and glomerular filtration rate in a group of Alport syndrome patients

Abstract Background and Aims Alport syndrome (AS) is the most common cause of inherited chronic kidney disease. This disorder is caused by deleterious variants on COL4A3, COL4A4 or COL4A5 genes. These genes codify the proteins that constitute collagen type IV of the glomerular basement membrane (GBM). Alterations in any of collagen type IV constituents cause disruption of the GMB structure, allowing the leakage of red blood cells and proteins, mainly albumin, into the urine resulting in kidney damage and loss of renal function. In AS progressive kidney impairment can be delayed or possibly prevented by timely initiation of renin angiotensin aldosterone inhibitors (RAAi): in males with X-linked AS and in autosomal recessive AS ARAS at diagnosis and in females with XLAS and autosomal dominant AS at the onset of microalbuminuria. Recently, it was showed that sodium-cotransporter-2 inhibitors (SGLT2i) reduce proteinuria and slow the progression of kidney disease in many glomerular diseases. Thus, also in AS, SGLT2i could be promising in renal disease progression control. The aim of this study was to determine the proteinuria reduction and the effect on the glomerular filtration rate (GFR) in a group of AS patients with persistent proteinuria besides the use of RAAi in maximum tolerated doses after adding SGLT2i to the treatment. Method This study was single-center, observational and prospective study evolving 8 AS adult patients, without a diagnosis of diabetes, with persistent proteinuria besides the use of RAAi in maximum tolerated doses. All patients were prescribed wit dapagliflozin 10 mg daily. We determine the changes from baseline of urinary protein/creatine (Up/cr) and glomerular filtration rate (based on epiGFR formula). Results Eight AS patients were included in this study. All were male patients. One patient has XLAS and the other 7 patients have ADAS. No patient was diabetic, and all were under RAAi in maximum tolerated doses. We determine de Up/cr and epiGFR at baseline and median time of 213.5 days (IQR 208.3-259) after SGLT2i initiation. At baseline, the median of Up/cr was 1.4 (IQR 0.8-2.1) and after SGLT2i Up/cr was 1.5 (0.8-2.6), p = 0.727. Up/cr, after SGLT2i treatment, reduced in 3 patients and worsened in 5. Median epiGFR at baseline was 80.3 ml/min (IQR 40.9-109.5) and after SGLT2i initiation was 74.7 ml/min (40.4-112.0), p = 0.727. Conclusion In this study, we did not observe an improvement in Up/cr of AS patients with SGLT2i treatment. A slight decrease in GFR was observed that may be due to the well know hemodynamic effects of SGLT2i. The small cohort and short time of the study are factors that can interfere in these results and should not discourage the treatment of AS patients with SGLT2i.

Genetic heterogeneity in epilepsy and comorbidities: insights from Pakistani families

BackgroundEpilepsy, a challenging neurological condition, is often present with comorbidities that significantly impact diagnosis and management. In the Pakistani population, where financial limitations and geographical challenges hinder access to advanced diagnostic methods, understanding the genetic underpinnings of epilepsy and its associated conditions becomes crucial.MethodsThis study investigated four distinct Pakistani families, each presenting with epilepsy and a spectrum of comorbidities, using a combination of whole exome sequencing (WES) and Sanger sequencing. The epileptic patients were prescribed multiple antiseizure medications (ASMs), yet their seizures persist, indicating the challenging nature of ASM-resistant epilepsy.ResultsIdentified genetic variants contributed to a diverse range of clinical phenotypes. In the family 1, which presented with epilepsy, developmental delay (DD), sleep disturbance, and aggressive behavior, a homozygous splice site variant, c.1339–6 C > T, in the COL18A1 gene was detected. The family 2 exhibited epilepsy, intellectual disability (ID), DD, and anxiety phenotypes, a homozygous missense variant, c.344T > A (p. Val115Glu), in the UFSP2 gene was identified. In family 3, which displayed epilepsy, ataxia, ID, DD, and speech impediment, a novel homozygous frameshift variant, c.1926_1941del (p. Tyr643MetfsX2), in the ZFYVE26 gene was found. Lastly, family 4 was presented with epilepsy, ID, DD, deafness, drooling, speech impediment, hypotonia, and a weak cry. A homozygous missense variant, c.1208 C > A (p. Ala403Glu), in the ATP13A2 gene was identified.ConclusionThis study highlights the genetic heterogeneity in ASM-resistant epilepsy and comorbidities among Pakistani families, emphasizing the importance of genotype-phenotype correlation and the necessity for expanded genetic testing in complex clinical cases.

Upregulation of collagen type X alpha 1promotes the progress of triple-negative breast cancer via Wnt/β-catenin signaling.

Triple-negative breast cancer (TNBC) is a malignant tumor with high degree of malignancy and lack of effective target treatment. The research aims to explore the role and mechanism of X collagen alpha-1 chain protein (COL10A1 gene) in TNBC. UALCAN and Kaplan-Meier were used to detect the expression of COL10A1 and its role in the prognosis of breast cancer patients. The cells with stably expressing high levels of COL10A1 were obtained by recombinant lentivirus infection. The expression of COL10A1 in cells was temporarily downregulated by siRNA interference fragments. Real-time quantitative polymerase chain reaction and western blot analysis were utilized to detect the changes of COL10A1 mRNA and protein expression. The biological functions of the cells were evaluated by colony formation, cell counting kit-8, cell invasion and wound healing experiments. In addition, the effect of COL10A1 on angiogenesis was investigated by tube formation assay. Xenograft tumor model was used to confirm the effect of COL10A1 on tumorigenicity in vivo and multiplex fluorescent immunohistochemistry to detect multiple proteins simultaneously. The possible molecular mechanism of the function of COL10A1 was speculated through the detection of proteins in functionally related pathways. COL10A1 is highly expressed and is significantly associated with worse overall survival (OS) and recurrence-free survival (RFS) in TNBC. Overexpression of COL10A1 increased the clone formation rate and cell migration capacity of TNBC cells. In the COL10A1 overexpression group, the clone formation rates of MD-MB-231 and BT-549 cells (21.5 ± 0.62, 27.83 ± 3.72)% were significantly higher than those in the control group(15.23 ± 2.79, 19.4 ± 1.47)%, and the relative migration ratio (47.40 ± 3.09, 41.26 ± 4.33)% were higher than those in the control group (34.48 ± 2.03, 21.80 ± 1.03)%. When the expression of COL10A1 was downregulated, the ability of clone formation and wound-healing migration capacity in TNBC cells was weakened. Upregulated COL10A1 in TNBC cells generated more junctions and longer total segments between vascular endothelial cells, and promoted angiogenesis of the cells, and thus enhanced the tumorigenesis. In TNBC, it was found that COL10A1 might affect epithelial-mesenchymal transition (EMT) of the cells through Wnt/β-catenin signaling pathway by the detection of the related pathway proteins. COL10A1 is highly expressed in TNBC, and its high expression leads to poor OS and RFS. COL10A1 may enhance TNBC cell proliferation, migration and tumor-related angiogenesis, and promote tumorigenesis in vivo via Wnt/β-catenin signaling.

#3084 Familial nephropathy associated with a combined mutations of Alport and Nail Patella syndrome genes in the same family

Abstract Introduction Alport syndrome is a genetic condition stemming from mutation in Type IV collagen and is characterized by kidney disease and frequently hearing loss and ocular abnormalities. Renal involvement is from an abnormality of the glomerular basement membrane with hematuria and proteinuria progressing eventually to renal failure. In the vast majority, the inheritance is X- linked affecting the alpha 5 chain of type IV collagen while in about 15 percent of the patients the inheritance is autosomal recessive or dominant with mutations affecting alpha 3 or 4 chains of type IV collagen. Sensorineural deafness is common and ocular abnormalities may include lenticonus, corneal opacities, fleck retinopathy and temporal retinal thinning. Nail Patella syndrome (NPS) is a rare genetic condition from mutation in the LMX1B gene on chromosome 9 with an autosomal dominant inheritance. Glomerular disease is from characteristic glomerular basement membrane changes and is associated with nail, pelvic girdle and limb abnormalities. A forme fruste with glomerular lesions but lacking extrarenal features is recognized but uncommon. We present a family with mutations in both COL4A5 and LMX1B presenting with hematuria and proteinuria Case Presentation A 23 year old female was evaluated for hematuria and proteinuria. Her history was notable for kidney biopsy at the age thirteen showing focal and segmental glomerulosclerosis (FSGS). She was treated with tacrolimus for four years from age thirteen through seventeen. Patient was reportedly diagnosed with Nail Patella Syndrome after genetic testing. Her family history was notable for hematuria in her brother and hematuria and proteinuria in her mother. On physical examination she was noted to have no nail or skeletal abnormalities associated with Nail Patella Syndrome. Her labs were notable for a serum creatinine of 0.6 mg/dl and proteinuria of 155 mg/g creatinine of proteinuria. Urine microscopy revealed dysmorphic hematuria. Radiological imaging did not show skeletal abnormalities observed in Nail Patella Syndrome but genetic testing identified a pathogenic variant in COL4A5 c.919G>A (p.Gly307Ser) in addition to a variant of uncertain significance in LMX1B c.349G>A (p.Gly117Ser). Her brother aged 20 had a creatinine of 0.87 mg/dl with dysmorphic hematuria and no proteinuria. Genetic testing showed the same pathogenic variant in COL4A5 as in his sibling. His mother aged 55 was subsequently evaluated and had a creatinine 0.73 mg/dl with dysmorphic hematuria and 152 mg/g of proteinuria. Genetic testing was with the same variant in COL4A5 noted in her two children. Renal biopsy was extensive thinning and segmental lamellation of the glomerular basement membrane with eight percent FSGS. The typical “moth-eaten” appearance of the glomerular basement membrane characteristic of NPS was not evident. Segmental staining was observed with alpha 5 unit of Type IV collagen. Audiology and retinal optical coherence optometry studies are pending. Discussion Alport syndrome and Nail Patella Syndrome are rare genetic disorders that cause variable degrees of renal insufficiency from glomerular base membrane defects and are associated with characteristic extra renal manifestations. This is the first report of an inherited nephritis with combined mutations in COL4A5 and LMX1B. The clinical features and renal biopsy are compatible with Alport Syndrome and the significance of the mutation in LMX1B is uncertain. Conclusion This is the first case report of a combined mutation in COL4A5 and LMX1B gene presenting with hematuria and minimal proteinuria with preserved kidney function.

Induction of Human Wharton's Jelly of Umbilical Cord Derived Mesenchymal Stem Cells to Be Chondrocytes and Transplantation in Guinea Pig Model with Spontaneous Osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease commonly found in elderly people and obese patients. Currently, OA treatments are determined based on their condition severity and a medical professional's advice. The aim of this study was to differentiate human Wharton's jelly-derived mesenchymal stem cells (hWJ-MSCs) into chondrocytes for transplantation in OA-suffering guinea pigs. hWJ-MSCs were isolated using the explant culture method, and then, their proliferation, phenotypes, and differentiation ability were evaluated. Subsequently, hWJ-MSCs-derived chondrocytes were induced and characterized based on immunofluorescent staining, qPCR, and immunoblotting techniques. Then, early-OA-suffering guinea pigs were injected with hyaluronic acid (HA) containing either MSCs or 14-day-old hWJ-MSCs-derived chondrocytes. Results showed that hWJ-MSCs-derived chondrocytes expressed specific markers of chondrocytes including Aggrecan, type II collagen, and type X collagen proteins and β-catenin, Sox9, Runx2, Col2a1, Col10a1, and ACAN gene expression markers. Administration of HA plus hWJ-MSCs-derived chondrocytes (HA-CHON) produced a better recovery rate of degenerative cartilages than HA plus MSCs or only HA. Histological assessments demonstrated no significant difference in Mankin's scores of recovered cartilages between HA-CHON-treated guinea pigs and normal articular cartilage guinea pigs. Transplantation of hWJ-MSCs-derived chondrocytes was more effective than undifferentiated hWJ-MSCs or hyaluronic acid for OA treatment in guinea pigs. This study provides a promising treatment to be used in early OA patients to promote recovery and prevent disease progression to severe osteoarthritis.

Genome-wide identification and analysis of epithelial-mesenchymal transition-related RNA-binding proteins and alternative splicing in a human breast cancer cell line

Exploring the mechanism of breast cancer metastasis and searching for new drug therapeutic targets are still the focuses of current research. RNA-binding proteins (RBPs) may affect breast cancer metastasis by regulating alternative splicing (AS) during epithelial-mesenchymal transition (EMT). We hypothesised that during EMT development in breast cancer cells, the expression level of RBPs and the gene AS pattern in the cell were significantly changed on a genome-wide scale. Using GEO database, this study identified differentially expressed RBPs and differential AS events at different stages of EMT in breast cancer cells. By establishing the correlation network of differential RBPs and differential AS events, we found that RBM47, PCBP3, FRG1, SRP72, RBMS3 and other RBPs may regulate the AS of ITGA6, ADGRE5, TNC, COL6A3 and other cell adhesion genes. By further analysing above EMT-related RBPs and AS in breast cancer tissues in TCGA, it was found that the expression levels of ADAT2, C2orf15, SRP72, PAICS, RBMS3, APOBEC3G, NOA1, ACO1 and the AS of TNC and COL6A3 were significantly correlated with the prognosis of breast cancer patients. The expression levels of all 8 RBPs were significantly different in breast cancer tissues without metastasis compared with normal breast tissues. Conclusively, eight RBPs such as RBMS3 and AS of TNC and COL6A3 could be used as predictors of breast cancer prognosis. These findings need to be further explored as possible targets for breast cancer treatment.

#2161 Phosphodiesterase V inhibitor attenuates the fibrotic gene expression of human renal fibroblast derived from IgA nephropathy: an in-vitro study

Abstract Background and Aims Renal fibrosis (RF) is the final common pathway that determines the progression of many glomerular disease including IgA nephropathy (IgAN). Till date there is no definite therapy available to retard RF. Being the key mediator of fibrosis, TGF-β is being targeted in many studies. Phosphodiesterase V inhibitor (PDE5i) is known to inhibit TGF-β but its role in RF is yet to be established. Hence, in the current in-vitro study we wish to establish the role of PDE5 inhibitor as an inhibitor of TGF-β and hence the RF. Method This is a pilot study to evaluate the efficacy of ‘Phosphodiesterase V inhibitor’ on TGFβ1 induced fibrosis on fibroblasts derived from biopsy specimen of IgA nephropathy patients. Renal fibroblasts were cultured from six IgA patients. The fibroblasts cells were treated with PDE5i inhibitor (tadalafil) (at different doses of 1 µm, 5 µm, 10 µm and 15 µm), both before and after stimulation with transforming growth factor beta-1 (TGF-b1) (at doses of 5 ng/ml, 10 ng/ml, 15 ng/ml and 20 ng/ml). Gene expression was studied by real-time polymerase chain reaction (RT-PCR) for messenger ribonucleic acid (mRNA) of pro-fibrotic genes Col1a1, Col1a2, FN1, CTGF, ASMA, TIMP1, and anti-fibrotic MMP2. Objectives Results Our results shows that TGFβ significantly increased the expression of profibrotic gene (Col1a1, Col1a2, ASMA, CTGF, FN and TIMP1) at concentration of 10 ng/ml in the fibroblast cells derived from IgAN patients. Treatment of fibroblast cells with Tadalafil (PDE5 inhibitor) significantly decreased the pro-fibrotic gene mRNA expression (Col1a1, Col1a2, ASMA, CTGF, FN and TIMP1) with increased anti-fibrotic gene expression (MMP2) at the dose of 5 µm and 10 µm concentration in the fibroblast cells (Fig. 1). Conclusion This study may provide potential therapeutic target for prevention of renal fibrosis in IgAN patients. Till date PDE5 inhibitors were not used for IgAN. We found that, PDE5 inhibitor reduces the fibrosis in this in vitro study, this drug may be proposed as a potential therapy for IgAN.

Sericin promotes chondrogenic proliferation and differentiation via glycolysis and Smad2/3 TGF-β signaling inductions and alleviates inflammation in three-dimensional models

Knee osteoarthritis is a chronic joint disease mainly characterized by cartilage degeneration. The treatment is challenging due to the lack of blood vessels and nerve supplies in cartilaginous tissue, causing a prominent limitation of regenerative capacity. Hence, we investigated the cellular promotional and anti-inflammatory effects of sericin, Bombyx mori-derived protein, on three-dimensional chondrogenic ATDC5 cell models. The results revealed that a high concentration of sericin promoted chondrogenic proliferation and differentiation and enhanced matrix production through the increment of glycosaminoglycans, COL2A1, COL X, and ALP expressions. SOX-9 and COL2A1 gene expressions were notably elevated in sericin treatment. The proteomic analysis demonstrated the upregulation of phosphoglycerate mutase 1 and triosephosphate isomerase, a glycolytic enzyme member, reflecting the proliferative enhancement of sericin. The differentiation capacity of sericin was indicated by the increased expressions of procollagen12a1, collagen10a1, rab1A, periostin, galectin-1, and collagen6a3 proteins. Sericin influenced the differentiation capacity via the TGF-β signaling pathway by upregulating Smad2 and Smad3 while downregulating Smad1, BMP2, and BMP4. Importantly, sericin exhibited an anti-inflammatory effect by reducing IL-1β, TNF-α, and MMP-1 expressions and accelerating COL2A1 production in the early inflammatory stage. In conclusion, sericin demonstrates potential in promoting chondrogenic proliferation and differentiation, enhancing cartilaginous matrix synthesis through glycolysis and TGF-β signaling pathways, and exhibiting anti-inflammatory properties.

A recurrent de novo missense mutation in COL1A1 causes osteogenesis imperfecta type II and preterm delivery in Normande cattle

BackgroundNine male and eight female calves born to a Normande artificial insemination bull named “Ly” were referred to the French National Observatory of Bovine Abnormalities for multiple fractures, shortened gestation, and stillbirth or perinatal mortality.ResultsUsing Illumina BovineSNP50 array genotypes from affected calves and 84 half-sib controls, the associated locus was mapped to a 6.5-Mb interval on chromosome 19, assuming autosomal inheritance with germline mosaicism. Subsequent comparison of the whole-genome sequences of one case and 5116 control genomes, followed by genotyping in the affected pedigree, identified a de novo missense substitution within the NC1 domain of the COL1A1 gene (Chr19 g.36,473,965G > A; p.D1412N) as unique candidate variant. Interestingly, the affected residue was completely conserved among 243 vertebrate orthologs, and the same substitution in humans has been reported to cause type II osteogenesis imperfecta (OI), a connective tissue disorder that is characterized primarily by bone deformity and fragility. Moreover, three COL1A1 mutations have been described to cause the same syndrome in cattle. Necropsy, computed tomography, radiology, and histology confirmed the diagnosis of type II OI, further supporting the causality of this variant. In addition, a detailed analysis of gestation length and perinatal mortality in 1387 offspring of Ly and more than 160,000 progeny of 63 control bulls allowed us to statistically confirm in a large pedigree the association between type II OI and preterm delivery, which is probably due to premature rupture of fetal membranes and has been reported in several isolated cases of type II OI in humans and cattle. Finally, analysis of perinatal mortality rates and segregation distortion supported a low level of germ cell mosaicism in Ly, with an estimate of 4.5% to 7.7% of mutant sperm and thus 63 to 107 affected calves born. These numbers contrast with the 17 cases reported and raise concerns about the underreporting of congenital defects to heredo-surveillance platforms, even for textbook genetic syndromes.ConclusionsIn conclusion, we describe a large animal model for a recurrent substitution in COL1A1 that is responsible for type II OI in humans. More generally, this study highlights the utility of such datasets and large half-sib families available in livestock species to characterize sporadic genetic defects.

The NM_033380.2 transcript of the COL4A5 gene contains a variable splice site c.4822-10T>C, which has been identified as a causative factor for Alport syndrome.

Alport Syndrome (AS) is a genetic kidney disorder characterized by progressive hearing loss and atypical eye symptoms, resulting in a poor prognosis and lack of effective targeted therapy. The primary mode of inheritance is X-linked dominant (XLAS) due to variants in the COL4A5 gene. This study revealed a previously unidentified alternative form of the COL4A5 gene, namely, the c.4822-10T>C variant, which was confirmed through in vitro experiments. To investigate the impact of a splicing variant on COL4A5 mRNA production, an in vitro minigene splicing assay was utilized. Additionally, molecular dynamics was employed to predict the ability of α5(IV) to form a triple helix. Results from the experiment revealed that the wild-type (WT) plasmid produced two distinct mRNA products simultaneously. Sequence analysis using the BLAST database revealed a 173-bp deletion in the mRNA sequence of the first product, indicating a potential similarity to the XM_016942897.2 transcript of Pan troglodytes. The second mRNA product of the WT plasmid contained the full sequence of exons 51, 52, and 53, as anticipated. Conversely, the mutant (MT) plasmid generated a single mRNA product with a 173-bp deletion in exon 52, leading to the identification of the mature mRNA expression as NM_033380.2: COL4A5: c.4822_4994del. In the context of nonsense-mediated mRNA decay (NMD), the deletion c.4822_4994 results in the production of a truncated protein, p.His1608*, that terminates prematurely. This truncated protein may disrupt the secondary structure of α5(IV) and potentially cause an abnormal conformation of α345(IV). This study examines the relationship between the variable splicing pattern in the NM_033380.2 transcript of the COL4A5 gene in XLAS patients and the presence of the COL4A5 gene splice variant c.4822-10T>C. Our findings indicate that the c.4822-10T>C splice variant leads to activation of nonsense-mediated mRNA degradation (NMD) and reduced COL4A5 mRNA expression, resulting in inadequate synthesis of the corresponding proteins. This aligns with the patient's immunofluorescence results showing negative α5(IV) chain presence at the glomerular basement membrane, bursa, and tubular basement membrane, confirming the pathogenic nature of c.4822-10T>C.

Identification of seven variants in the col4a1 gene that alter RNA splicing by minigene assay.

Type IV collagen is an integral component of basement membranes. Mutations in COL4A1, one of the key genes encoding Type IV collagen, can result in a variety of diseases. It is clear that a significant proportion of mutations that affect splicing can cause disease directly or contribute to the susceptibility or severity of disease. Here, we analyzed exonic mutations and intronic mutations described in the COL4A1 gene using bioinformatics programs and identified candidate mutations that may alter the normal splicing pattern through a minigene system. We identified seven variants that induce splicing alterations by disrupting normal splice sites, creating new ones, or altering splice regulatory elements. These mutations are predicted to impact protein function. Our results help in the correct molecular characterization of variants in COL4A1 and may help develop more personalized treatment options.

Systems biology approach: identification of hub genes, signaling pathways, and molecular docking of COL1A1 gene in cervical insufficiency

Systems biology approach: identification of hub genes, signaling pathways, and molecular docking of COL1A1 gene in cervical insufficiency

Galunisertib downregulates mutant type I collagen expression and promotes MSCs osteogenesis in pediatric osteogenesis imperfecta

Qualitative alterations in type I collagen due to pathogenic variants in the COL1A1 or COL1A2 genes, result in moderate and severe Osteogenesis Imperfecta (OI), a rare disease characterized by bone fragility. The TGF-β signaling pathway is overactive in OI patients and certain OI mouse models, and inhibition of TGF-β through anti-TGF-β monoclonal antibody therapy in phase I clinical trials in OI adults is rendering encouraging results. However, the impact of TGF-β inhibition on osteogenic differentiation of mesenchymal stem cells from OI patients (OI-MSCs) is unknown. The following study demonstrates that pediatric skeletal OI-MSCs have imbalanced osteogenesis favoring the osteogenic commitment. Galunisertib, a small molecule inhibitor (SMI) that targets the TGF-β receptor I (TβRI), favored the final osteogenic maturation of OI-MSCs. Mechanistically, galunisertib downregulated type I collagen expression in OI-MSCs, with greater impact on mutant type I collagen, and concomitantly, modulated the expression of unfolded protein response (UPR) and autophagy markers. In vivo, galunisertib improved trabecular bone parameters only in female oim/oim mice. These results further suggest that type I collagen is a tunable target within the bone ECM that deserves investigation and that the SMI, galunisertib, is a promising new candidate for the anti-TGF-β targeting for the treatment of OI.

Prenatal phenotype and genetic analysis of a fetus with Fibrochondrogenesis 1 due to compound heterozygous variants of COL11A1 gene

To explore the genetic etiology of a fetus with short limbs identified by prenatal ultrasonography. A fetus detected with short limb malformations at Shengjing Hospital Affiliated to China Medical University on October 25, 2021 was selected as the study subject. Prenatal ultrasound and post-abortion imaging were carried out to determine the phenotypic characteristics of the fetus. Amniotic fluid sample of the fetus and peripheral blood samples of its parents were collected. Following extraction of genomic DNA, whole-exome sequencing was carried out. Candidate variants were verified by Sanger sequencing. Online software was used to predict the structural changes of the mutant proteins. Prenatal ultrasound showed that the fetus had a small bell-shaped thorax, markedly shortened limbs, flat midface, a small nose with anteriorly tilted nostrils, and a small mandible. Post-abortion CT showed typical short and wide fetal ribs, cupped metaphyses at both ends, short long bones with wide metaphyses, resulting in a dumbbell-shaped appearance and curved thoracic vertebrae. Whole-exome sequencing revealed that the fetus had harbored compound heterozygous variants of the COL11A1 gene, namely c.2251G>T and c.3790G>T, both of which were predicted to alter the important Gly-X-Y structure of collagen protein. Sanger sequencing confirmed that the variants were respectively inherited from its parents. A rare fetus with Fibrochondrogenesis type 1 due to compound heterozygous variants of the COL11A1 gene has been diagnosed. Above finding has enabled genetic counseling and reproductive guidance for this family.

Creation and characterization of novel rat model for recessive dystrophic epidermolysis bullosa: Frameshift mutation of the Col7a1 gene leads to severe blistered phenotype.

Recessive dystrophic epidermolysis bullosa is a rare genodermatosis caused by a mutation of the Col7a1 gene. The Col7a1 gene codes for collagen type VII protein, a major component of anchoring fibrils. Mutations of the Col7a1 gene can cause aberrant collagen type VII formation, causing an associated lack or absence of anchoring fibrils. This presents clinically as chronic blistering, scarring, and fibrosis, often leading to the development of cutaneous squamous cell carcinoma. Patients also experience persistent pain and pruritus. Pain management and supportive bandaging remain the primary treatment options. The pathology of recessive dystrophic epidermolysis bullosa was first described in the 1980s, and there has since been a multitude of encouraging treatment options developed. However, in vivo research has been hindered by inadequate models of the disease. The various mouse models in existence possess longevity and surface area constraints, or do not adequately model a normal human disease state. In this paper, we describe a novel rat model of recessive dystrophic epidermolysis bullosa that offers an alternative to previous murine models. An 8-base pair deletion was induced in the Col7a1 gene of Lewis rats, which was subsequently found to cause a premature stop codon downstream. Homozygous mutants presented with a fragile and chronically blistered phenotype postnatally. Further histological analysis revealed subepidermal clefting and the absence of anchoring fibrils. The generation of this novel model offers researchers an easily maintained organism that possesses a larger surface area for experimental topical and transfused therapies to be tested, which may provide great utility in the future study of this debilitating disease.

A comprehensive analysis of metabolomics and lipidomics in areca nut mediated oral submucous fibrosis progression through LCMS and Raman spectroscopy

AbstractHabitual consumption of areca/betel nut in Southeast Asia has been associated with oral submucous fibrosis (OSMF) and its malignant transformation to oral squamous cell carcinoma. The current study aimed to assess the molecular alterations in fibroblast cell lines after treatment with areca nut. Areca nut extract (ANE) was prepared and characterized for its active ingredient, arecoline. ANE‐treated cells were subjected to cell viability, proliferation, migration, morphologic changes, and transcript of OSMF genes (col1a1, col1a2, hsp47, timp1, timp2, timp3, and timp4). Further, liquid chromatography–mass spectrometry (LCMS) of cell lysate and Raman microspectroscopy (RMS) of fixed cells were performed for metabolomics/lipidomics and biomolecular alterations in the nucleus and periphery of ANE‐treated cells. We compared and integrated the data from both techniques and observed that the cells treated with ANE mimicked OSMF and could be used as an in vitro model. LCMS showed a significant alteration in 17 metabolites and 165 lipid molecules in the cells treated with ANE. Further, 40 molecules in the nucleus and 29 in the periphery were found to be altered in these cells when subjected to RMS. Molecules associated with pathways such as the transfer of acetyl groups into mitochondria, amino sugar metabolism, and the Warburg effect were modulated the most upon ANE treatment. Acetyl CoA was found to be common in most of the altered pathways. Besides, the pathways affecting carbohydrate metabolism were also altered significantly. Targeting these molecules and pathways can help to prevent the malignant transformation of OSMF.

A novel combination therapy targets sonic hedgehog signaling by the dual inhibition of HMG-CoA reductase and HSP90 in rats with non-alcoholic steatohepatitis

Non-alcoholic steatohepatitis (NASH) is characterized by liver inflammation, fat accumulation, and collagen deposition. Due to the limited availability of effective treatments, there is a pressing need to develop innovative strategies. Given the complex nature of the disease, employing combination approaches is essential. Hedgehog signaling has been recognized as potentially promoting NASH, and cholesterol can influence this signaling by modifying the conformation of PTCH1 and SMO activity. HSP90 plays a role in the stability of SMO and GLI proteins. We revealed significant positive correlations between Hedgehog signaling proteins (Shh, SMO, GLI1, and GLI2) and both cholesterol and HSP90 levels. Herein, we investigated the novel combination of the cholesterol-lowering agent lovastatin and the HSP90 inhibitor PU-H71 in vitro and in vivo. The combination demonstrated a synergy score of 15.09 and an MSA score of 22.85, as estimated by the ZIP synergy model based on growth inhibition rates in HepG2 cells. In a NASH rat model induced by thioacetamide and a high-fat diet, this combination therapy extended survival, improved liver function and histology, and enhanced antioxidant defense. Additionally, the combination exhibited anti-inflammatory and anti-fibrotic potential by influencing the levels of TNF-α, TGF-β, TIMP-1, and PDGF-BB. This effect was evident in the suppression of the Col1a1 gene expression and the levels of hydroxyproline and α-SMA. These favorable outcomes may be attributed to the combination's potential to inhibit key Hedgehog signaling molecules. In conclusion, exploring the applicability of this combination contributes to a more comprehensive understanding and improved management of NASH and other fibrotic disorders.

Development of CRISPR/Cas9-Based Mouse Models for X-linked Alport Syndrome

Intro: Alport Syndrome (AS) is a rare hereditary disorder characterized by progressive renal impairment and sensorineural hearing loss, resulting from mutations in the genes that encode COL4 synthesis. X-linked Alport Syndrome (XLAS) is the most common form of AS, comprising an estimated 80% of all human AS cases (the others being various forms of autosomal recessive or autosomal dominant AS), and is caused by various mutations on the COL4A5 gene. However, to date, there are no animal models of XLAS. Methods: To facilitate comprehensive preclinical investigations and therapeutic developments, we established two novel mouse models of XLAS using CRISPR/Cas9 technology. Leveraging the robust C57Bl/6J mouse strain, we precisely introduced the L1655R and C1570S mutations, which represent the predominant human variants of XLAS observed in the Western United States. Discussion: Our innovative mouse models not only serve as invaluable tools for elucidating the underlying mechanisms of XLAS pathogenesis but also provide a platform for evaluating the effcacy of potential therapeutic interventions. The establishment of these faithful genetic replicas of human XLAS marks a significant advancement in the field, offering unprecedented opportunities for in-depth mechanistic studies and the development of targeted therapeutic strategies for this debilitating renal and auditory disorder. This research is supported by the National Institutes of Health under Ruth L. Kirschstein National Research Service Award 5T32DK091317 from the National Institute of Diabetes and Digestive and Kidney Diseases and the National Kidney Foundation of Utah and Idaho. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Characterization of cardiac fibrosis in older lean vs. obese male ZSF1 rats

The ZSF1 rat is a novel strain produced by crossing a ZDF (Zucker diabetic fatty) female and SHHF (Spontaneously Hypertensive Heart Failure) male rat. Offspring of this hybrid have two phenotypes: healthy lean or obese prone to diabetes and heart failure. The metabolic syndrome and hypertension displayed by the obese ZSF1 rat mimics that seen in individuals who are predisposed to heart failure with preserved ejection fraction (HFpEF). Historically, HFpEF is a diffcult disease to accurately model with rodents and the ZSF1 rat is a promising model for the spontaneous progression to HFpEF. In recent studies, the oldest age at which ZSF1 rats were studied was 32 weeks and these obese rats displayed diastolic dysfunction, cardiac fibrosis, and hypertrophy. Since the prevalence of HFpEF increases with advanced age, the aim of the current study is to evaluate the extent of fibrosis in older ZSF1 rats (40-42 weeks) and determine whether this age group is more appropriate for the study of fibrotic cardiac remodeling related to HFpEF. We hypothesize that left ventricular (LV) fibrosis and collagen gene expression will be increased in obese compared to lean counterparts. Furthermore, we expect that the extent of fibrosis in this age group will exceed that reported in 32-week-old rats; thus, providing an improved model for the study of fibrotic heart disease. Lean and obese male ZSF1 rats (n=8/group) were aged to 40-42 weeks. Rats were anesthetized with a ketamine/xylazine cocktail and cardiac function was evaluated by invasive hemodynamics. The heart was removed, and the LV was trimmed and weighed. A mid-myocardial section from the LV was fixed and embedded in paraffn for histology. Remaining LV tissue was frozen for extraction of RNA and protein for biochemical analysis. LV collagen was assessed by Sirius red stain and collagen I ( Col1a1) and III ( Col3a1) gene expression were assessed by RTqPCR. With no significant difference in heart rate, obese ZSF1 rats had higher LV developed pressure compared to lean (191.1 vs. 122.0 mmHg, p<0.05) and increased maximum and minimum dP/dT (p<0.05). Obese ZSF1 rats exhibited cardiac hypertrophy as measured by LV weight normalized to tibial length (30.21 vs. 23.61 mg/mm, p<0.05, n=7/group) and increased fibrosis (Sirius red) compared to lean counterparts. Furthermore, the fibrosis in obese ZSF1 rats was concentrated in the endocardial and perivascular regions, and qualitatively more than previously published in the 32-week-old ZSF1. Preliminarily, obese ZSF1 rats exhibit a 3.5-fold increase in Col1a1 (p=0.02) and a 19-fold increase in C ol3a1 (p=0.06) gene expression compared to lean (n=3/group). Interestingly, not only is this a larger increase than that reported in 32-week-old old ZSF1s, but the relative increase in Col3a1 vs. Col1a1 is larger. Taken together, these data suggest a progression of cardiac fibrosis beyond what has been reported in the 32-week-old ZSF1 rats. Use of aged ZSF1 rats may be valuable for the study of fibrogenic mediators in HFpEF for the elucidation of therapeutic agents to reduce or reverse cardiac fibrosis. Future studies will include female rats to evaluate sex-specific progression of fibrosis. North Carolina Biotechnology Center Flash Grant (JH) and ECU REDE startup program (AG). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.