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Trimerization and Genotype-Phenotype Correlation of COL4A5 Mutants in Alport Syndrome.

ABSTRACT: Introduction:Alport syndrome is a hereditary glomerulonephritis that results from the disruption of collagen ?345(IV) heterotrimerization caused by mutation in COL4A3, COL4A4 or COL4A5 genes. Many clinical studies have elucidated the correlation between genotype and phenotype, but there is still much ambiguity and insufficiency. Here, we focused on the ?345(IV) heterotrimerization of ?5(IV) missense mutant as a novel factor to further understand the pathophysiology of Alport syndrome. Methods:We selected 9 ?5(IV) missense mutants with typical glycine substitutions that clinically differed in disease progression. To quantify the trimerization of each mutant, split nanoluciferase-fused ?3/?5 mutants and ?4 were transfected into the cells, and intracellular and secreted heterotrimer were detected by luminescence using an assay that we developed previously. Results:Trimer formation and secretion patterns tended to be similar to the wild type in most of the mutations that did not show proteinuria at a young age. On the other hand, trimer secretion was significantly reduced in all the mutations that showed proteinuria and early onset of renal failure. One of these mutants has low ability of intracellular trimer formation, and the others had the defect of low-level secretion. In addition, the mutant that is assumed to be nonpathogenic has similar trimer formation and secretion pattern as wild-type ?5(IV). Conclusion:The result of cell-based ?345(IV) heterotrimer formation assay was largely correlated with clinical genotype-phenotype. These trimerization assessments provide additional phenotypic considerations and may help to distinguish between pathogenic and nonpathogenic mutations.

SUBMITTER: Kamura M

PROVIDER: S-EPMC7210609 | biostudies-literature | 2020 May

REPOSITORIES: biostudies-literature

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Trimerization and Genotype-Phenotype Correlation of <i>COL4A5</i> Mutants in Alport Syndrome.

Kamura Misato M Yamamura Tomohiko T Omachi Kohei K Suico Mary Ann MA Nozu Kandai K Kaseda Shota S Kuwazuru Jun J Shuto Tsuyoshi T Iijima Kazumoto K Kai Hirofumi H

Kidney international reports 20200130 5

<h4>Introduction</h4>Alport syndrome is a hereditary glomerulonephritis that results from the disruption of collagen α345(IV) heterotrimerization caused by mutation in <i>COL4A3</i>, <i>COL4A4</i> or <i>COL4A5</i> genes. Many clinical studies have elucidated the correlation between genotype and phenotype, but there is still much ambiguity and insufficiency. Here, we focused on the α345(IV) heterotrimerization of α5(IV) missense mutant as a novel factor to further understand the pathophysiology o ...[more]

PMID: 32405592

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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

Trimerization and Genotype-Phenotype Correlation of COL4A5 Mutants in Alport Syndrome.

Publications

Trimerization and Genotype-Phenotype Correlation of <i>COL4A5</i> Mutants in Alport Syndrome.

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