Development of an exon skipping therapy for X-linked Alport syndrome with truncating variants in COL4A5.
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ABSTRACT:
SUBMITTER: Yamamura T
PROVIDER: S-EPMC7265383 | biostudies-literature |
REPOSITORIES: biostudies-literature
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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.
| S-EPMC7434753 | biostudies-literature
Project description:Background and objectivesX-linked Alport syndrome is caused by mutations in the COL4A5 gene. Although many COL4A5 mutations have been detected, the mutation detection rate has been unsatisfactory. Some men with X-linked Alport syndrome show a relatively mild phenotype, but molecular basis investigations have rarely been conducted to clarify the underlying mechanism.Design, setting, participants, & measurementsIn total, 152 patients with X-linked Alport syndrome who were suspected of having Alport syndrome through clinical and pathologic investigations and referred to the hospital for mutational analysis between January of 2006 and January of 2013 were genetically diagnosed. Among those patients, 22 patients had suspected splice site mutations. Transcripts are routinely examined when suspected splice site mutations for abnormal transcripts are detected; 11 of them showed expected exon skipping, but others showed aberrant splicing patterns. The mutation detection strategy had two steps: (1) genomic DNA analysis using PCR and direct sequencing and (2) mRNA analysis using RT-PCR to detect RNA processing abnormalities.ResultsSix splicing consensus site mutations resulting in aberrant splicing patterns, one exonic mutation leading to exon skipping, and four deep intronic mutations producing cryptic splice site activation were identified. Interestingly, one case produced a cryptic splice site with a single nucleotide substitution in the deep intron that led to intronic exonization containing a stop codon; however, the patient showed a clearly milder phenotype for X-linked Alport syndrome in men with a truncating mutation. mRNA extracted from the kidney showed both normal and abnormal transcripts, with the normal transcript resulting in the milder phenotype. This novel mechanism leads to mild clinical characteristics.ConclusionsThis report highlights the importance of analyzing transcripts to enhance the mutation detection rate and provides insight into genotype-phenotype correlations. This approach can clarify the cause of atypically mild phenotypes in X-linked Alport syndrome.
| S-EPMC4220764 | biostudies-literature
Project description:X-linked Alport syndrome (XLAS) is caused by pathogenic variants in COL4A5 and is characterized by progressive kidney disease, hearing loss, and ocular abnormalities. Recent advances in genetic analysis and further understanding of genotype-phenotype correlations in affected male patients raises the importance of detecting splicing variants in COL4A5. Aberrant splicing of COL4A5 is caused not only by canonical splice site variants but also non-canonical splice site variants such as deep intronic changes or even substitutions in exons. Patients with splicing variants account for ~15% of all cases in XLAS. In addition, it has been shown that there is a significant difference in kidney survival depending on the aberrant splicing patterns of transcripts- in particular in-frame or out-of-frame nucleotide changes in transcripts. Therefore, cDNA analysis of patient mRNA is necessary to determine the impact of splice site variants and to confirm a diagnosis of XLAS and to predict the kidney prognosis. However, it is usually difficult to amplify COL4A5 transcripts extracted from peripheral blood leukocytes. For these cases, in vitro minigene assays or RNA sequence extracted from urine derived cells can confirm aberrant splicing patterns. Moreover, controlling aberrant splicing by nucleic acids or small molecular compounds in genetic diseases are attracting attention as a potential therapeutic strategy. Here, we review the frequency of splicing variants in COL4A5, the latest diagnostic strategies, and the prospects for new therapeutic approaches.
| S-EPMC8861460 | biostudies-literature
Project description:Alport syndrome is an inherited chronic human kidney disease, characterized by glomerular basement membrane abnormalities. This disease is caused by mutations in COL4A3, COL4A4, or COL4A5 gene. The knockout mice for Col4α3, Col4α4, and Col4α5 are developed and well characterized for the study of Alport syndrome. However, disease progression and effects of pharmacological therapy depend on the genetic variability. This model was reliable only to mouse. In this study, we created a novel Alport syndrome rat model utilizing the rGONAD technology, which generated rat with a deletion of the Col4α5 gene. Col4α5 deficient rats showed hematuria, proteinuria, high levels of BUN, Cre, and then died at 18 to 28 weeks of age (Hemizygous mutant males). Histological and ultrastructural analyses displayed the abnormalities including parietal cell hyperplasia, mesangial sclerosis, and interstitial fibrosis. Then, we demonstrated that α3/α4/α5 (IV) and α5/α5/α6 (IV) chains of type IV collagen disrupted in Col4α5 deficient rats. Thus, Col4α5 mutant rat is a reliable candidate for the Alport syndrome model for underlying the mechanism of kidney diseases and further identifying potential therapeutic targets for human renal diseases.
| S-EPMC8531394 | biostudies-literature
Project description: Not available
| S-EPMC8575944 | biostudies-literature
Project description:IntroductionWomen and girls with X-linked Alport syndrome have a risk of disease progression that is difficult to predict. This systematic review examined whether proteinuria correlated with genotype and disease severity in this population.MethodsPubMed and Scopus were searched for manuscripts from the past 20 years with "COL4A5," "female," "proteinuria" and related terms. Genotypes and clinical data for women and girls with pathogenic heterozygous COL4A5 variants were extracted. Features were then compared between females with proteinuria or without proteinuria; and genotype-phenotype correlations for age at proteinuria and kidney failure determined.ResultsThree-hundred sixty-six women and girls with COL4A5 variants and a median age of 29 years (interquartile range 15-46) were identified. Eighty-eight (24%) had large rearrangements or truncating variants, 63 (17%) had splicing variants, and 215 (59%) had missense changes. In all, 236 (64%) had proteinuria, 56 (16%) had kidney failure, 40 (16%) had a hearing loss, and 15 (7%) had ocular abnormalities. Women and girls with proteinuria were more likely to have large rearrangements or truncating variants (P = 0.005), and less likely to have missense changes (P = 0.0002). Those with proteinuria were also more likely to develop kidney failure (P < 0.0001). Women and girls with truncating, large or splicing variants developed proteinuria earlier than those with missense changes (P = 0.001, P < 0.0001 respectively). Those whose proteinuria was detected before the age of 15 progressed to kidney failure sooner (P < 0.0001).ConclusionProteinuria correlates with a more severe genotype in women and girls with X-linked Alport syndrome and is an indicator of disease severity and likely progression.
| S-EPMC9751687 | biostudies-literature
Project description:Alport syndrome is the commonest inherited kidney disease and nearly half the pathogenic variants in the COL4A3-COL4A5 genes that cause Alport syndrome result in Gly substitutions. This study examined the molecular characteristics of Gly substitutions that determine the severity of clinical features. Pathogenic COL4A5 variants affecting Gly in the Leiden Open Variation Database in males with X-linked Alport syndrome were correlated with age at kidney failure (n = 157) and hearing loss diagnosis (n = 80). Heterozygous pathogenic COL4A3 and COL4A4 variants affecting Gly (n = 304) in autosomal dominant Alport syndrome were correlated with the risk of haematuria in the UK 100,000 Genomes Project. Gly substitutions were stratified by exon location (1 to 20 or 21 to carboxyl terminus), being adjacent to a non-collagenous region (interruption or terminus), and the degree of instability caused by the replacement residue. Pathogenic COL4A5 variants that resulted in a Gly substitution with a highly destabilising residue reduced the median age at kidney failure by 7 years (p = 0.002), and age at hearing loss diagnosis by 21 years (p = 0.004). Substitutions adjacent to a non-collagenous region delayed kidney failure by 19 years (p = 0.014). Heterozygous pathogenic COL4A3 and COL4A4 variants that resulted in a Gly substitution with a highly destabilising residue (Arg, Val, Glu, Asp, Trp) were associated with an increased risk of haematuria (p = 0.018), and those adjacent to a non-collagenous region were associated with a reduced risk (p = 0.046). Exon location had no effect. In addition, COL4A5 variants adjacent to non-collagenous regions were over-represented in the normal population in gnomAD (p < 0.001). The nature of the substitution and of nearby residues determine the risk of haematuria, early onset kidney failure and hearing loss for Gly substitutions in X-linked and autosomal dominant Alport syndrome.
| S-EPMC8854626 | biostudies-literature
Project description: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.
| S-EPMC6295608 | biostudies-literature
Project description: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.
| S-EPMC10436629 | biostudies-literature
Project description:Background 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. Methods 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. Results 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. Conclusions Our present finding expands the mutational spectrum for the COL4A5 gene associated with XLAS and highlights the genotype-phenotype correlations in this disease.
| S-EPMC8506736 | biostudies-literature