Establishment of X-linked Alport syndrome model mice with a Col4a5 R471X mutation.
ABSTRACT: Alport syndrome (AS) is an inherited disorder characterized by glomerular basement membrane (GBM) abnormality and development of chronic kidney disease at an early age. The cause of AS is a genetic mutation in type IV collagen, and more than 80% of patients have X-linked AS (XLAS) with mutation in COL4A5. Although the causal gene has been identified, mechanisms of progression have not been elucidated, and no effective treatment has been developed. In this study, we generated a Col4a5 mutant mouse harboring a nonsense mutation (R471X) obtained from a patient with XLAS using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated system. Col4a5 mRNA and protein expressions were not observed in the kidneys of hemizygous R471X male mice. R471X mice showed proteinuria and hematuria. Pathology revealed progression of glomerulosclerosis and interstitial fibrosis by age. Electron microscopy identified irregular thickening in GBM accompanied by irregular lamination. These observations were consistent with the clinical and pathological features of patients with AS and other established models. In addition, our mice models develop end-stage renal disease at the median age of 28 weeks, much later compared to previous models much more consistent with clinical course of human XLAS. Our models have advantages for future experiments in regard with treatment for human XLAS.
Project description:X-linked Alport syndrome (XLAS) is a progressive kidney disease caused by genetic abnormalities of COL4A5. Lack of collagen IV ?5 chain staining and "basket-weave" by electron microscopy (EM) in glomerular basement membrane (GBM) are its typical pathology. However, the causal relationship between GBM defects and progressive nephropathy is unknown. We analyzed sequential pathology in a mouse model of XLAS harboring a human nonsense mutation of COL4A5. In mutant mice, nephropathy commenced from focal GBM irregularity by EM at 6 weeks of age, prior to exclusive crescents at 13 weeks of age. Low-vacuum scanning EM demonstrated substantial ragged features in GBM, and crescents were closely associated with fibrinoid exudate, despite lack of GBM break and podocyte depletion at 13 weeks of age. Crescents were derived from two sites by different cellular components. One was CD44?+?cells, often with fibrinoid exudate in the urinary space, and the other was accumulation of ?-SMA?+?cells in the thickened Bowman's capsule. These changes finally coalesced, leading to global obliteration. In conclusion, vulnerability of glomerular and capsular barriers to the structural defect in collagen IV may cause non-necrotizing crescents via activation of PECs and migration of interstitial fibroblasts, promoting kidney disease in this model.
Project description:<h4>Background</h4> Chronic kidney disease caused by X-linked Alport syndrome (XLAS) is relatively rare. However, due to the nonspecific pathologic and clinical manifestations of this disease, it is easily misdiagnosed. Genetic testing is crucial in identifying suspected cases. In addition, the results of genetic testing are an important indicator of patient prognosis. This study demonstrated a novel pathogenic COL4A5 splicing site variant in a Chinese family with XLAS. <h4>Methods</h4> Targeted next generation sequencing (NGS) was performed to identify the gene variant in the family members, and the gene mutation site was confirmed by Sanger sequencing. We then further analyzed the consequences of this gene mutation on the translated protein of this variant using in silico and in vitro approaches. <h4>Results</h4> A novel splice region variant, c.1033-2(IVS 18) A>G, in COL4A5 intron 18 was identified in the affected family members. Sanger sequencing confirmed that this variant is segregated with disease. In silico analysis, this variant led to frame-shift and premature termination on the translation of the nucleic acid, and this result was verified by RNA splicing analysis in a cell model. Unexpectedly, we still observed positive immunohistology staining of collagen IV α5 in the glomerular basement membrane (GBM) of the index patient, which implied that another potential transcription or translation mechanism skipping the mutated site might exist. <h4>Conclusions</h4> Our present finding expands the mutational spectrum for the COL4A5 gene associated with XLAS and highlights the genotype-phenotype correlations in this disease.
Project description:<h4>Background</h4>X-linked Alport syndrome (XLAS), caused by mutations in the type IV collagen COL4A5 gene, accounts for approximately 80% of human Alport syndrome. Dogs with XLAS have a similar clinical progression. Prior studies in autosomal recessive Alport mice demonstrated early mesangial cell invasion as the source of laminin 211 in the glomerular basement membrane (GBM), leading to proinflammatory signaling. The objective of this study was to verify this process in XLAS dogs.<h4>Methods</h4>XLAS dogs and WT littermates were monitored with serial clinicopathologic data and kidney biopsies. Biopsies were obtained at set milestones defined by the onset of microalbuminuria (MA), overt proteinuria, onset of azotemia, moderate azotemia, and euthanasia. Kidney biopsies were analyzed by histopathology, immunohistochemistry, and electron microscopy.<h4>Results</h4>XLAS dogs showed progressive decrease in renal function and progressive increase in interstitial fibrosis and glomerulosclerosis (based on light microscopy and immunostaining for fibronectin). The only identifiable structural abnormality at the time of microalbuminuria was ultrastructural evidence of mild segmental GBM multilamination, which was more extensive when overt proteinuria developed. Co-localization studies showed that mesangial laminin 211 and integrin ?8?1 accumulated in the GBM at the onset of overt proteinuria and coincided with ultrastructural evidence of mild cellular interpositioning, consistent with invasion of the capillary loops by mesangial cell processes.<h4>Conclusion</h4>In a large animal model, the induction of mesangial filopodial invasion of the glomerular capillary loop leading to the irregular deposition of laminin 211 is an early initiating event in Alport glomerular pathology.
Project description:Optical coherence tomography (OCT) findings of temporal macular thinning are important in the diagnosis and prognosis of X-linked Alport syndrome (XLAS).To report OCT findings and severity of temporal macular thinning in a cohort with XLAS and to correlate these and other ocular findings with mutation genotype.Patients with XLAS underwent genotyping for COL4A5 mutations and complete eye examinations with retinal imaging using spectral domain OCT and fundus photography. Temporal macular thinning was calculated from OCT measurements by comparing the ratio of the retinal thickness of the temporal to the nasal subfields with a published normative database.University departments of ophthalmology and nephrology.Thirty-two patients from 24 families.Temporal thinning index calculated from spectral domain OCT scans.All study patients had a mutation associated with the X-linked COL4A5 gene. Eleven different mutations were identified. Eleven of 32 patients (34%) expressed the L1649R mutation. Of a total of 63 eyes with available OCT scans, 44 (70%) had severe pathological temporal macular thinning. The L1649R mutation was associated with the least amount of severe temporal macular thinning and later onset of renal failure.Temporal macular thinning is a prominent sign associated with XLAS, suggesting that OCT measurements are essential in the diagnosis and prognosis of the disease. The L1649R mutation in the COL4A5 gene causes a relatively mild form of XLAS characterized by late-onset renal failure and less frequent, severe temporal macular thinning relative to other COL4A5 mutations. The pathological basis for the retinal abnormalities of XLAS remains to be established.
Project description:Currently, there are no treatments for Alport syndrome, which is the second most commonly inherited kidney disease. Here we report the development of an exon-skipping therapy using an antisense-oligonucleotide (ASO) for severe male X-linked Alport syndrome (XLAS). We targeted truncating variants in exon 21 of the COL4A5 gene and conducted a type IV collagen α3/α4/α5 chain triple helix formation assay, and in vitro and in vivo treatment efficacy evaluation. We show that exon skipping enabled trimer formation, leading to remarkable clinical and pathological improvements including expression of the α5 chain on glomerular and the tubular basement membrane. In addition, the survival period was clearly prolonged in the ASO treated mice group. This data suggests that exon skipping may represent a promising therapeutic approach for treating severe male XLAS cases.
Project description:BACKGROUND:Alport syndrome is an inherited renal disease caused by mutations in COL4A3, COL4A4, or COL4A5 genes. Coexisting mutations in either two of the three genes in Alport patients have been reported recently. However, the effect of heterozygous mutations in COL4A3 or COL4A4 genes in X-linked Alport syndrome (XLAS) patients is unclear. METHODS:Using targeted next-generation sequencing, six unrelated Chinese children were identified to have a combination of a pathogenic variant in COL4A5 and a heterozygous mutation in COL4A3 or COL4A4. They were three males and three females. Another three XLAS males each with only one pathogenic variant in COL4A5 were included. The clinical data were analyzed and compared between the males in two groups (group 1, males with a pathogenic variant in COL4A5 and a heterozygous pathogenic variant in COL4A3 or COL4A4; group 2, males with only one pathogenic variant in COL4A5). RESULTS:Patients with XLAS who also had heterozygous pathogenic COL4A3 or COL4A4 variants accounted for 1% of Alport syndrome. In this study, three children showed coexisting pathogenic variants in COL4A5 and COL4A3. Two children showed pathogenic variants in COL4A5 and COL4A4. One child had pathogenic variants in the three COL4A3-5 genes, in which the pathogenic variant in COL4A5 was de novo and the pathogenic variants in COL4A4 and COL4A3 were inherited independently (in trans). The site and type of mutations in COL4A5 were similar between the two groups. It was revealed that males in group 1 presented more severe proteinuria than males in group 2 (p < 0.05). CONCLUSION:The present study provides further evidence for complicated genotype in Alport syndrome. For the first time, we reported a case with three pathogenic variants in COL4A5, COL4A3, and COL4A4 genes. Moreover, we found that heterozygous pathogenic COL4A3 or COL4A4 variants are likely to make XLAS disease more serious.
Project description:Alport syndrome is a hereditary progressive chronic kidney disease caused by mutations in type IV collagen genes COL4A3/4/5. X-linked Alport syndrome (XLAS) is caused by mutations in the COL4A5 gene and is the most common form of Alport syndrome. A strong correlation between the type of COL4A5 mutation and the age developing end-stage renal disease in male patients has been found. Mutation to the ? (IV) chain causes retention of the protein to the endoplasmic reticulum lumen, which causes endoplasmic reticulum stress (ERS) and subsequent exertion of deleterious intracellular effects through the activation of ERS. The exact time point that mutant type IV collagen ? chain exerts its deleterious effects remains elusive. In this study, we explored the relationship between the COL4A5 genotype and cell type in ERS activation. We obtained skin fibroblasts from Alport syndrome patients with different COL4A5 mutation categories [i.e., a missense mutation (c.4298G > T, p.Gly1433Val) in exon 47, a splicing mutation (c.1949-1G > A) in intron 25 and an insertion (c.573_c.574insG, p. Pro193Alafs*23) in exon 10], and then reprogrammed these fibroblasts into induced pluripotent stem cells (iPSCs). Interestingly, no significant dysregulation of ERS pathway markers was observed for the three COL4A5 mutant iPSCs; however, significant activation of ERS in COL4A5 mutant fibroblasts was observed. In addition, we found that the activation levels of some ERS markers in fibroblasts varied among the three COL4A5 mutation types. Mutant COL4A5 proteins were demonstrated to have different effects on cells at different stages of ontogenesis, providing a theoretical basis for choosing the timing of intervention. The observed differences in activation of ERS by the COL4A5 mutant fibroblasts may contribute to the intracellular molecular mechanisms that describe the correlation between genotype and clinical features in XLAS.
Project description:BACKGROUND:X-linked Alport syndrome (XLAS) is a progressive, hereditary glomerular nephritis of variable severity caused by pathogenic COL4A5 variants. Currently, genetic testing is widely used for diagnosing XLAS; however, determining the pathogenicity of variants detected by such analyses can be difficult. Intronic variants or synonymous variants may cause inherited diseases by inducing aberrant splicing. Transcript analysis is necessary to confirm the pathogenicity of such variants, but it is sometimes difficult to extract mRNA directly from patient specimens. METHODS:In this study, we conducted in vitro splicing analysis using a hybrid minigene assay and specimens from three XLAS patients with synonymous variants causing aberrant splicing, including previously reported pathogenic mutations in the same codon. The variants were c.876 A>T (p.Gly292=), c.2358 A>G (p.Pro786=), and c.3906 A>G (p.Gln1302=). RESULTS:The results from our hybrid minigene assay were sufficient to predict splicing abnormalities; c.876 A>T cause 17-bp del and 35-bp del, c.2358 A>G cause exon 29 skipping, c.3906 A>G cause exon 42 skipping, which are very likely to cause pathogenicity. Further, patients carrying c.2358 A>G exhibited a mild phenotype that may have been associated with the presence of both normal and abnormally spliced transcripts. CONCLUSION:The minigene system was shown to be a sensitive assay and a useful tool for investigating the pathogenicity of synonymous variants.
Project description:<h4>Introduction</h4><i>COL4A5</i> is a causative gene of X-linked Alport syndrome (XLAS). Male patients with XLAS with nonsense variants have the most severe phenotypes of early onset end-stage kidney disease (ESKD); those with splicing variants have middle phenotypes and those with missense variants have the mildest phenotypes. Therefore, genotyping for male patients with XLAS can be used to predict kidney prognosis. Single-base substitutions at the last nucleotide position in each exon are known to affect splicing patterns and could be splicing variants. Nevertheless, in XLAS, these variants are generally considered to be missense variants, without conducting a transcript analysis, which underestimates some patients as having mild phenotypes. This study aimed to investigate whether single-base substitutions at the last nucleotide position of <i>COL4A5</i> exons cause aberrant splicing.<h4>Methods</h4>In total, 20 variants were found in the Human Gene Mutation Database (<i>n</i> = 14) and our cohort (<i>n</i> = 6). We performed functional splicing assays using a hybrid minigene analysis and <i>in vivo</i> transcript analyses of patients' samples when available. Then, we investigated genotype-phenotype correlations for patients with splicing variants detected in this study by comparing data from our previous studies.<h4>Results</h4>Among the 20 variants, 17 (85%) caused aberrant splicing. Male patients with splicing variants had more severe phenotypes when compared with those with missense variants. Findings from the <i>in vivo</i> analyses for 3 variants were identical to those from the minigene assay.<h4>Conclusion</h4>Our study revealed that most single-base substitutions at the last nucleotide position of <i>COL4A5</i> exons result in splicing variants, rather than missense variants, thereby leading to more severe phenotypes.
Project description:X-linked Alport syndrome (XLAS) is an inherited renal disease caused by mutations in COL4A5 gene. The c.2858G>T(p.(G953V)) in COL4A5 gene (rs78972735) has been considered pathogenic previously. However, there are conflicting interpretations of its pathogenicity recently. Here we presented 19 Chinese families, out of which 36 individuals (18 probands and 18 family members) carried the c.2858G>T(p.(G953V)) in COL4A5 gene. The clinical manifestations and genetic findings of them were analyzed. We found there were no clinical features of Alport syndrome not only in six probands with c.2858G>T(p.(G953V)) in COL4A5 plus pathogenic variants in other genes (e.g., WT1, ADCK4, NPHP1, TRPC6, COL4A4, and PAX2) but also in another six probands with only the c.2858G>T(p.(G953V)) variant. The other six probands with a combination of c.2858G>T(p.(G953V)) and another pathogenic variant in COL4A5 had XLAS. Eleven family members (11/18, nine females and two males) who had only the c.2858G>T(p.(G953V)) variant were asymptomatic. These two males (at age of 42 and 35 years) had normal result of urine analysis and no more clinical traits of Alport syndrome. We conclude c.2858G>T(p.(G953V)) in COL4A5 gene is not a pathogenic variant for XLAS. Individuals should not be diagnosed as XLAS only based on the detection of c.2858G>T(p.(G953V)) in COL4A5 gene.