COL7A1 Mutations Research Articles

A Current Landscape on Alport Syndrome Cases: Characterization, Therapy and Management Perspectives.

Alport syndrome (AS) is a rare genetic disorder categorized by the progressive loss of kidney function, sensorineural hearing loss and eye abnormalities. It occurs due to mutations in three genes that encode for the alpha chains of type IV collagen. Globally, the disease is classified based on the pattern of inheritance into X-linked AS (XLAS), which is caused by pathogenic variants in COL4A5, representing 80% of AS. Autosomal recessive AS (ARAS), caused by mutations in either COL4A3 or COL4A4, represents 15% of AS. Autosomal dominant AS (ADAS) is rare and has been recorded in 5% of all cases due to mutations in COL4A3 or COL4A4. This review provides updated knowledge about AS including its clinical and genetic characteristics in addition to available therapies that only slow the progression of the disease. It also focuses on reported cases in Saudi Arabia and their prevalence. Moreover, we shed light on advances in genetic technologies like gene editing using CRISPR/Cas9 technology, the need for an early diagnosis of AS and managing the progression of the disease. Eventually, we provide a few recommendations for disease management, particularly in regions like Saudi Arabia where consanguineous marriages increase the risk.

Clinical and molecular features in a cohort of Middle Eastern patients with epidermolysis bullosa.

Epidermolysis bullosa (EB) features skin and mucosal fragility due to pathogenic variants in genes encoding components of the cutaneous basement membrane. Based on the level of separation within the dermal-epidermal junction, EB is sub-classified into four major types including EB simplex (EBS), junctional EB (JEB), dystrophic EB (DEB), and Kindler EB (KEB) with 16 EB-associated genes reported to date. We ascertained a cohort of 151 EB patients of various Middle Eastern ethnic backgrounds. The cohort was comprised of EBS (64%, 97/151), DEB (21%, 31/151), JEB (12%, 18/151), and KEB (3%, 5/151). KRT14 and KRT5 variants were most common among EBS patients with 43% (42/97) and 46% (45/97) of EBS patients carrying mutations in either of these two genes, respectively. Truncal involvement was more common in KRT14-associated EBS as compared to EBS due to KRT5 mutations (p < .05). Mutations in COL17A1 and laminin 332-encoding genes were identified in 55% (10/18) and 45% (8/18) of JEB patients. Scarring alopecia, caries, and EB nevi were most common among JEB patients carrying COL17A1 mutations as compared to laminin 332-associated JEB (p < .05). Abnormal nails were evident in most DEB and JEB patients while poikiloderma was exclusively observed in KEB (p < .001). EB patients of Middle Eastern origin were found to feature specific phenotype-genotype correlations of relevance to the diagnosis and genetic counseling of patients in this region.

Analysis of miRNAs in Osteogenesis imperfecta Caused by Mutations in COL1A1 and COL1A2: Insights into Molecular Mechanisms and Potential Therapeutic Targets.

Osteogenesis imperfecta (OI) is a group of connective tissue disorders leading to abnormal bone formation, mainly due to mutations in genes encoding collagen type I (Col I). Osteogenesis is regulated by a number of molecules, including microRNAs (miRNAs), indicating their potential as targets for OI therapy. The goal of this study was to identify and analyze the expression profiles of miRNAs involved in bone extracellular matrix (ECM) regulation in patients diagnosed with OI type I caused by mutations in COL1A1 or COL1A2. Primary skin fibroblast cultures were used for DNA purification and sequence analysis, followed by analysis of miRNA expression. Sequencing analysis revealed mutations of the COL1A1 or COL1A2 genes in all OI patients, including four previously unreported. Amongst the 40 miRNAs analyzed, 9 were identified exclusively in OI cells and 26 in both OI patients and the controls. In the latter case, the expression of six miRNAs (hsa-miR-10b-5p, hsa-miR-19a-3p, hsa-miR-19b-3p, has-miR-204-5p, has-miR-216a-5p, and hsa-miR-449a) increased, while four (hsa-miR-129-5p, hsa-miR-199b-5p, hsa-miR-664a-5p, and hsa-miR-30a-5p) decreased significantly in OI cells in comparison to their expression in the control cells. The identified mutations and miRNA expression profiles shed light on the intricate processes governing bone formation and ECM regulation, paving the way for further research and potential therapeutic advancements in OI and other genetic diseases related to bone abnormality management.

Epidermolysis bullosa with col7a1 mutation at Jiangsu university hospital: A Case report and review of the literature

We describe a case of a new born Chinese male baby who was diagnosed with Epidermolysis Bullosa (EB), a rare genetic skin illness that causes particular blisters to appear on the baby's lower limbs immediately after delivery. The flaw results from the skin's reduced ability to adhere to supporting tissue, which makes it brittle. Through carrier mother inheritance, genetic testing identified our case as an EB patient with Aplasia Cutis Congenita after a nucleotide alteration in the COL7A1 gene.

OP-03 Osteogenesis Imperfecta Type 3 with COL1A1 mutation Prenatal diagnosis and management

OP-03 Osteogenesis Imperfecta Type 3 with COL1A1 mutation Prenatal diagnosis and management

IPSC-derived type IV collagen α5-expressing kidney organoids model Alport syndrome

Alport syndrome (AS) is a hereditary glomerulonephritis caused by COL4A3, COL4A4 or COL4A5 gene mutations and characterized by abnormalities of glomerular basement membranes (GBMs). Due to a lack of curative treatments, the condition proceeds to end-stage renal disease even in adolescents. Hampering drug discovery is the absence of effective in vitro methods for testing the restoration of normal GBMs. Here, we aimed to develop kidney organoid models from AS patient iPSCs for this purpose. We established iPSC-derived collagen α5(IV)-expressing kidney organoids and confirmed that kidney organoids from COL4A5 mutation-corrected iPSCs restore collagen α5(IV) protein expression. Importantly, our model recapitulates the differences in collagen composition between iPSC-derived kidney organoids from mild and severe AS cases. Furthermore, we demonstrate that a chemical chaperone, 4-phenyl butyric acid, has the potential to correct GBM abnormalities in kidney organoids showing mild AS phenotypes. This iPSC-derived kidney organoid model will contribute to drug discovery for AS.

Combination of osteogenesis imperfecta and hypophosphatasia in three children with multiple fractures, low bone mass and severe osteomalacia, a challenge for therapeutic management

Osteogenesis imperfecta (OI) and hypophosphatasia (HPP) are rare skeletal disorders caused by mutations in the genes encoding collagen type I (COL1A, COL1A2) and tissue-non-specific isoenzyme of alkaline phosphatase (ALPL), respectively. Both conditions result in skeletal deformities and bone fragility although bone tissue abnormalities differ considerably. Children with OI have low bone mass and hypermineralized matrix, whereas HPP children develop rickets and osteomalacia.We report a family, father and three children, affected with growth retardation, low bone mass and recurrent fractures. None of them had rickets, blue sclera or dentinogenesis imperfecta. ALP serum levels were low and genetics revealed in the four probands heterozygous pathogenic mutations in COL1A2 c.838G > A (p.Gly280Ser) and in ALPL c.1333T > C (p.Ser445Pro). After multidisciplinary meeting, a diagnostic transiliac bone biopsy was indicated for each sibling for therapeutic decision.Bone histology and histomorphometry, as compared to reference values of children with OI type I as well as, to a control pediatric patient harboring the same COL1A2 mutation, revealed similarly decreased trabecular bone volume, increased osteocyte lacunae, but additionally severe osteomalacia. Quantitative backscattered electron imaging demonstrated that bone matrix mineralization was not as decreased as expected for osteomalacia.In summary, we observed within each biopsy samples classical features of OI and classical features of HPP. The apparent nearly normal bone mineralization density distribution results presumably from divergent effects of OI and HPP on matrix mineralization. A combination therapy was initiated with ALP enzyme-replacement and one month later with bisphosphonates. The ongoing treatment led to improved skeletal growth, increased BMD and markedly reduced fracture incidence.

Scoliosis in osteogenesis imperfecta: identifying the genetic and non-genetic factors affecting severity and progression from longitudinal data of 290 patients

BackgroundScoliosis is widely prevalent among osteogenesis imperfecta (OI) patients, and is progressive with age. However, factors affecting scoliosis in OI are not well known.MethodsWe retrospectively retrieved longitudinal radiographic and clinical records of consecutive OI patients seeking treatments at our hospital from 2014 to 2022, graded their pre-operative spinal conditions into four outcome groups, estimated their progression rates, and descriptively and inferentially analyzed the genetic and non-genetic factors that may affect the outcomes and progression rates.ResultsIn all, 290 OI patients met the inclusion criteria, where 221 had genetic records. Of these 221, about 2/3 had mutations in COL1A1 or COL1A2, followed by mutations in WNT1 (9.0%), IFITM5 (9.0%) and other OI risk genes. With an average age of 12.0 years (interquartile range [IQR] 6.9–16.1), 70.7% of the cohort had scoliosis (Cobb angle > 10°), including 106 (36.5%) mild (10°–25°), 40 (13.8%) moderate (25°–50°), and 59 (20.3%) severe (> 50°) scoliosis patients. Patients with either COL1A1 and COL1A2 were strongly biased toward having mild or no scoliosis, whereas patients with mutations in IFITM5, WNT1 and other recessive genes were more evenly distributed among the four outcome grades. Lower-limb discrepancy, bone mineral density (BMD) and age of first drug used were all significantly correlated with severity outcomes. Using multivariate logistic regression, we estimated that each year older adds an odds ratio of 1.13 (95% confidence interval [CI] 1.07–1.2) in progression into advanced stages of scoliosis. We estimated a cohort-wide progression rate of 2.7 degrees per year (95% CI 2.4–3.0). Early-onset patients experienced fast progressions during both infantile and adolescent stages. Twenty-five of the 59 (42.8%) patients with severe scoliosis underwent spinal surgeries, enjoying an average Cobb angle reduction of 33° (IQR 23–40) postoperatively.ConclusionThe severity and progression of scoliosis in osteogenesis imperfecta were affected by genetic factors including genotypes and mutation types, and non-genetic factors including age and BMD. As compared with COL1A1, mutations in COL1A2 were less damaging while those on IFITM5 and other recessive genes conferred damaging effects. Progression rates were the fastest in the adolescent adult age-group.

Inflammation-mediated fibroblast activation and immune dysregulation in collagen VII-deficient skin.

Inflammation is known to play a critical role in all stages of tumorigenesis; however, less is known about how it predisposes the tissue microenvironment preceding tumor formation. Recessive dystrophic epidermolysis bullosa (RDEB), a skin-blistering disease secondary to COL7A1 mutations and associated with chronic wounding, inflammation, fibrosis, and cutaneous squamous cell carcinoma (cSCC), models this dynamic. Here, we used single-cell RNA sequencing (scRNAseq) to analyze gene expression patterns in skin cells from a mouse model of RDEB. We uncovered a complex landscape within the RDEB dermal microenvironment that exhibited altered metabolism, enhanced angiogenesis, hyperproliferative keratinocytes, infiltration and activation of immune cell populations, and inflammatory fibroblast priming. We demonstrated the presence of activated neutrophil and Langerhans cell subpopulations and elevated expression of PD-1 and PD-L1 in T cells and antigen-presenting cells, respectively. Unsupervised clustering within the fibroblast population further revealed two differentiation pathways in RDEB fibroblasts, one toward myofibroblasts and the other toward a phenotype that shares the characteristics of inflammatory fibroblast subsets in other inflammatory diseases as well as the IL-1-induced inflammatory cancer-associated fibroblasts (iCAFs) reported in various cancer types. Quantitation of inflammatory cytokines indicated dynamic waves of IL-1α, TGF-β1, TNF, IL-6, and IFN-γ concentrations, along with dermal NF-κB activation preceding JAK/STAT signaling. We further demonstrated the divergent and overlapping roles of these cytokines in inducing inflammatory phenotypes in RDEB patients as well as RDEB mouse-derived fibroblasts together with their healthy controls. In summary, our data have suggested a potential role of inflammation, driven by the chronic release of inflammatory cytokines such as IL-1, in creating an immune-suppressed dermal microenvironment that underlies RDEB disease progression.

Identification of a novel COL7A1 variant associated with dystrophic epidermolysis bullosa pruriginosa responding effectively to dupilumab.

Variants in COL7A1 cause an extremely rare and clinically heterogeneous syndrome known as dystrophic epidermolysis bullosa pruriginosa (DEB-Pr). Duplilumab, a fully humanized anti-IL-4Ra monoclonal antibody, can inhibit IL-4 and IL-13-driven signaling. Ethical Compliance: Following our Institutional Review Board, genetic testing has been made available after completing a signed informed consent form. This article presents the case study of a DEB-Pr patient who received dupilumab therapy. Genomic DNA was extracted from the peripheral blood of the patient. The findings showed that a unique COL7A1 mutation was discovered in the patient who underwent genetic testing. As a result of the patient receiving dupilumab treatment, the individual reported experiencing significantly less itching and considerably improved erythema, less severe scales, crusts, and flattening of plaques. In conclusion, the current investigation showed that to the best of our knowledge, this is the first DEB-Pr patient with heterozygous COL7A1 (NM_000094.3:c.8110G>A [p. Gly2704Arg]) who responded positively to dupilumab treatment without experiencing any serious side effects.

The UCMD-Causing COL6A1 ( c . 930 + 189 C &gt; T ) Intron Mutation Leads to the Secretion and Aggregation of Single Mutated Collagen VI α1 Chains

Collagen VI is a unique member of the collagen family. Its assembly is a complex multistep process and the vulnerability of the process is manifested in muscular diseases. Mutations in COL6A1, COL6A2, and COL6A3 lead to the severe Ullrich Congenital Muscular Dystrophy (UCMD) and a spectrum of disease of varying severity including the milder Bethlem muscular dystrophy. The recently identified dominant intronic mutation in COL6A1 ( c . 930 + 189 C &gt; T ) leads to the partial in-frame insertion of a pseudoexon between exon 11 and exon 12. The pseudoexon is translated into 24 amino acid residues in the N-terminal region of the triple helix and results in the interruption of the typical G-X-Y motif. This recurrent de novo mutation leads to UCMD with a severe progression within the first decade of life. Here, we demonstrate that a mutation-specific antibody detects the mutant chain colocalizing with wild type collagen VI in the endomysium in patient muscle. Surprisingly, in the cell culture of patient dermal fibroblasts, the mutant chain is secreted as a single α chain, while in parallel, normal collagen VI tetramers are assembled with the wild-type α1 chain. The mutant chain cannot be incorporated into collagen VI tetramers but forms large aggregates in the extracellular matrix that may retain the ability to interact with collagen VI and potentially with other molecules. Also, α1 chain-deficient WI-26 VA4 cells transfected with the mutant α1 chain do not assemble collagen VI tetramers but, instead, form aggregates. Interestingly, both the wild type and the mutant single α1 chains form amorphous aggregates when expressed in HEK293 cells in the absence of α2 and α3 chains. The detection of aggregated, assembly incompetent, mutant collagen VI α1 chains provides novel insights into the disease pathophysiology of UCMD patients with the COL6A1 ( c . 930 + 189 C &gt; T ) mutation.

A novel mutation in COL1A1 causing osteogenesis imperfecta/hearing loss

ObjectivesTo screen the COL1A1 and COL1A2 gene mutation sites in a family with type I osteogenesis imperfecta (OI)/hearing loss and analyze the characteristics and recovery of hearing loss in patients with osteogenesis imperfecta. MethodsThe basic clinical data of OI proband and her parents were collected, and the COL1A1 and COL1A2 genes were detected in peripheral blood by PCR amplification and generation Sanger sequencing. Literature of stapedial surgery in patients with osteogenesis imperfecta was collected. ResultsThe heterozygous mutation of the 26 exon c.1922_1923 ins C in the OI progenitor COL1A1 gene led to the amino acid frameshift mutation of p.Pro 601FS, which was not detected in the phenotypic parents. The homozygous of exon 28 c.1782>G in COL1A2 was detected in the proband and her parents, resulting in changes in the protein p.Pro 549Ala. ConclusionThe clinical symptoms of the OI proband is caused by heterozygous mutation of the 26 exon c.1922_1923 ins C in COL1A1 gene. Stapedial surgery can provide short-term and long-term hearing benefits for OI patients with hearing loss. Level of evidenceLevel 4.

Temporal Progression of Aortic Valve Pathogenesis in a Mouse Model of Osteogenesis Imperfecta.

Organization of extracellular matrix (ECM) components, including collagens, proteoglycans, and elastin, is essential for maintaining the structure and function of heart valves throughout life. Mutations in ECM genes cause connective tissue disorders, including Osteogenesis Imperfecta (OI), and progressive debilitating heart valve dysfunction is common in these patients. Despite this, effective treatment options are limited to end-stage interventions. Mice with a homozygous frameshift mutation in col1a2 serve as a murine model of OI (oim/oim), and therefore, they were used in this study to examine the pathobiology of aortic valve (AoV) disease in this patient population at structural, functional, and molecular levels. Temporal echocardiography of oim/oim mice revealed AoV dysfunction by the late stages of disease in 12-month-old mice. However, structural and proteomic changes were apparent much earlier, at 3 months of age, and were associated with disturbances in ECM homeostasis primarily related to collagen and proteoglycan abnormalities and disorganization. Together, findings from this study provide insights into the underpinnings of late onset AoV dysfunction in connective tissue disease patients that can be used for the development of mechanistic-based therapies administered early to halt progression, thereby avoiding late-stage surgical intervention.

Novel nonsense mutation in COL4A4 associated with Alport syndrome

Mutations are the major cause of genetic disorders. Development of next generation sequencing techniques have played a vital role in the identification of mutations in the patients which help us to relate specific mutations to various disorders. In an effort to identify and correlate mutations to Alport syndrome, whole exome sequencing and Sanger sequencing were carried out in a family having significant family history of Alport syndrome on maternal side. Since mutations in collagen encoding genes, especially COL4A3, COL4A4 and COL4A5 are known to cause Alport syndrome, various in-silico tools were used to screen for deleterious mutations in these genes. Here we report a novel mutation in COL4A4, Lys1688 to stop gain mutation in the patient. Analysis of the mutation using various databases revealed that this mutation occurs in a highly conserved region of COL4A4 protein and also it is predicted to be deleterious. Structural analysis and comparison of the mutant protein with wild type protein using in-silico tools also predict that the mutant protein has loss of function and could lead to the pathophysiology of the Alport syndrome.

Clinical features and morphology of collagen fibrils in patients with vascular Ehlers-Danlos based on electron microscopy.

Background: Vascular-type Ehlers-Danlos syndrome (vEDS) is caused by collagen III deficit resulting from heterogeneous mutations in COL3A1, which occasionally causes sudden death due to arterial/visceral rupture. However, it is difficult to conduct basic research on the pathophysiology of vEDS. Moreover, the number of patients with vEDS is small, limiting the number of available samples. Furthermore, the symptoms of vEDS may vary among family members, even if they share the same mutation. Accordingly, many aspects of the pathology of vEDS remain unknown. Therefore, we investigated the structural abnormalities in collagen fibrils and endoplasmic reticulum (ER) stress in skin samples using electron microscopy as well as their relationship with clinical symptoms in 30 patients with vEDS (vEDS group) and 48 patients without vEDS (disease-negative control group). Methods: Differences between the two groups were evaluated in terms of the sizes of collagen fibrils using coefficient of variation (COV). Results: COV was found to be significantly higher in the vEDS group than in the disease-negative control group, indicating irregularity in the size of collagen fibrils. However, in the vEDS group, some patients had low COV and seldom experienced serious complications and ER stress. Conclusion: ER stress might affect collagen fibril-composing proteins. Moreover, as this stress varies among people based on environmental factors and aging, it may be the underlying cause of varying vEDS symptoms.

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.

Percutaneous transjugular approach without arterial monitoring for the treatment of a direct carotid-cavernous fistula with vascular Ehlers-Danlos syndrome: illustrative case.

Vascular Ehlers-Danlos syndrome (vEDS) because of COL3A1 mutations is a rare inherited collagen vascular disease associated with spontaneous arterial dissections, aneurysms, vessel rupture, and organ rupture. A direct carotid-cavernous fistula (CCF) is the most common central nervous system vascular anomaly in vEDS; however, its treatment is challenging due to extremely fragile arteries and veins. A 22-year-old woman presented with pulsatile tinnitus and mild diplopia. CCF formation without trauma, cervical dissecting aneurysms, thin skin, and multiple ligament tears, as well as a genetic analysis, led to a diagnosis of vEDS. To minimize the risk of vascular injury in the thoracoperitoneal cavity, the internal jugular vein was directly punctured and the CCF was embolized transvenously using the triple-overlay road-mapping technique without arterial monitoring. The CCF was completely occluded, and the patient showed an excellent clinical course without neurological or vascular complications. Physicians and neurosurgeons should consider vEDS when treating younger patients with spontaneous CCF without trauma and investigate the possibility of genetic abnormalities and systemic vascular pathology. Transvenous embolization of a CCF through the transjugular route using the triple-overlay road-mapping technique can minimize the risk of vascular injury in a patient with vEDS.

P07 Optimizing DNA specificity and applicability of base and prime editors on COL7A1 variants causing recessive dystrophic epidermolysis bullosa

Abstract Base and prime editors are novel CRISPR variants that reduce the risk of off-target effects by avoiding double-stranded DNA cleavage, bypassing the need for homology-directed repair. These tools offer great therapeutic potential for inherited skin diseases such as recessive dystrophic epidermolysis bullosa (RDEB). Biallelic loss-of-function mutations in COL7A1 cause RDEB, resulting from loss of functional type VII collagen (C7) in the basement membrane zone. Current therapies for RDEB are limited, but ex vivo and in vivo COL7A1 base or prime editing therapies can potentially restore normal protein production with a single intervention treatment. This study compared DNA editing specificity, safety and efficiency on RDEB fibroblasts harbouring a homozygous c.4448 G&amp;gt;A (p.Gly1483Asp) mutation in COL7A1. Firstly, transfected adenine base editor (ABE8e) mRNA together with single guide RNA (sgRNA) were introduced into patient primary fibroblasts with Lipofectamine MessengerMax. By varying concentrations and ratios of guide RNA and ABE8e mRNA, we have achieved 100% biallelic correction without introducing any bystander mutations using 200 ng ABE8e and 28 ng sgRNA by transfecting 100 000 cells. Initial prime editing experiments are underway with PEmax plasmid and engineered prime editing guide RNAs (epegRNA) to optimize the guide design and to compare PE2max and PE4max editing systems. The functionality of the 100% biallelic correction using ABE8e is being evaluated with Western blot and in three-dimensional skin models. These early experiments demonstrate the potential value of base editing as a gene-editing tool in RDEB therapeutics; however, more functional work and off-target analyses are needed.

COL3A1 Mutations in Spontaneous Coronary Artery Dissection

COL3A1 Mutations in Spontaneous Coronary Artery Dissection

COL4A3 Mutation Induced Podocyte Apoptosis by Dysregulation of NADPH Oxidase 4 and MMP-2

Podocyte apoptosis is a common mechanism driving progression in Alport syndrome (AS). This study aimed to investigate the mechanism of podocyte apoptosis caused by COL4A3 mutations. We recruited patients with autosomal dominant AS (ADAS). Patients with minimal change disease (MCD) were recruited as controls. Microarray analysis was carried out on isolated glomeruli from the patients and validated. Then, corresponding mutant human podocytes (p.C1616Y) and 129 mice (p.C1615Y, the murine homolog to the human p.C1616Y) were constructed. The highest differentially expressed genes (DEGs) from microarray analysis were validated in transgenic mice and podocytes before and after administration of MMP-2 inhibitor (SB-3CT) and NOX4 inhibitor (GKT137831). We further validated NOX4/MMP-2/apoptosis pathway by real-time polymerase chain reaction (PCR), immunohistochemistry, and western blot in renal tissues from the ADAS patients. Using microarray analysis, we observed that DEGs, including NOX4/H2O2, MMP-2, and podocyte apoptosis-related genes were significantly upregulated. These genes were validated by real-time PCR, histologic analysis, and western blot in corresponding mutant human podocyte (p.C1616Y) and/or mice models (p.C1615Y). Moreover, we found podocyte apoptosis was abrogated and MMP-2 expression was down-regulated both invivo and invitro by NOX4 inhibition, urinary albumin-to-creatinine ratio, 24-hour proteinuria; and renal pathologic lesion was attenuated by NOX4 inhibition invivo. Furthermore, podocyte apoptosis was attenuated whereas NOX4 expression remained the same by inhibition of MMP-2 both invivo and invitro. These results indicated that NOX4 might induce podocyte apoptosis through the regulation of MMP-2 in patients with COL4A3 mutations. Our findings provided new insights into the mechanism of ADAS.