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A Drosophila model to screen Alport syndrome COL4A5 variants for their functional pathogenicity.

ABSTRACT: Alport syndrome is a hereditary chronic kidney disease, attributed to rare pathogenic variants in either of three collagen genes (COL4A3/4/5) with most localized in COL4A5. Trimeric type IV Collagen α3α4α5 is essential for the glomerular basement membrane that forms the kidney filtration barrier. A means to functionally assess the many candidate variants and determine pathogenicity is urgently needed. We used Drosophila, an established model for kidney disease, and identify Col4a1 as the functional homolog of human COL4A5 in the fly nephrocyte (equivalent of human podocyte). Fly nephrocytes deficient for Col4a1 showed an irregular and thickened basement membrane and significantly reduced nephrocyte filtration function. This phenotype was restored by expressing human reference (wildtype) COL4A5, but not by COL4A5 carrying any of three established pathogenic patient-derived variants. We then screened seven additional patient COL4A5 variants; their ClinVar classification was either likely pathogenic or of uncertain significance. The findings support pathogenicity for four of these variants; the three others were found benign. Thus, demonstrating the effectiveness of this Drosophila in vivo kidney platform in providing the urgently needed variant-level functional validation.

SUBMITTER: Duan J

PROVIDER: S-EPMC10979928 | biostudies-literature | 2024 Mar

REPOSITORIES: biostudies-literature

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A Drosophila model to screen Alport syndrome COL4A5 variants for their functional pathogenicity.

Duan Jianli J Wen Pei P Zhao Yunpo Y van de Leemput Joyce J Lai Yee Jennifer J Fermin Damian D Warady Bradley A BA Furth Susan L SL Ng Derek K DK Sampson Matthew G MG Han Zhe Z

bioRxiv : the preprint server for biology 20240311

Alport syndrome is a hereditary chronic kidney disease, attributed to rare pathogenic variants in either of three collagen genes (COL4A3/4/5) with most localized in COL4A5. Trimeric type IV Collagen α3α4α5 is essential for the glomerular basement membrane that forms the kidney filtration barrier. A means to functionally assess the many candidate variants and determine pathogenicity is urgently needed. We used Drosophila, an established model for kidney disease, and identify ...[more]

PMID: 38559272

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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.

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.

Dataset Information

A Drosophila model to screen Alport syndrome COL4A5 variants for their functional pathogenicity.

Publications

A <i>Drosophila</i> model to screen Alport syndrome <i>COL4A5</i> variants for their functional pathogenicity.

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