Dataset Information

Functional assessment of a novel COL4A5 splicing site variant in a Chinese X-linked Alport syndrome family

ABSTRACT:

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.

SUBMITTER: Chen X

PROVIDER: S-EPMC8506736 | biostudies-literature |

REPOSITORIES: biostudies-literature

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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.

Project description:Introduction: Alport syndrome (AS; OMIM#308940) is a progressive hereditary kidney disease characterized by hearing loss and ocular abnormalities. According to the mode of inheritance, AS has three subtypes: X-linked (XL; OMIM#301050), autosomal recessive (AR; OMIM#203780), and autosomal dominant (AD; OMIM#104200). XLAS is caused by a pathogenic variant in COL4A5 (OMIM*303630) gene encoding type IV collagen (Col-IV) α5 chain, while ADAS and ARAS are consequences of a variant in COL4A3 (OMIM*120070) and COL4A4 (OMIM*120131) genes that encode Col-IV α3 and α4 chains, respectively. Usually, diagnosis of AS requires hereditary or pathological examinations. Splicing variants are hard to be determined as pathogenic or non-pathogenic based on the results of gene sequencing. Methods: This study focused on a splicing variant in COL4A5 gene, termed NM_000495.5: c.4298-20T>A, and to analyzed its authenticity and damaged α5 chain. In vitro minigene splicing assay was applied to investigate the effect of splicing variant, c.4298-20T>A, on COL4A5 mRNA synthesis. Molecular dynamics method was used to predict the capability of the responsible α5(IV) to form a triple helix. Results: The intron 46 of COL4A5 mRNA retained 18 bp, resulting in insertion of six amino acids behind the amino acid at position 1,433 of α5(IV). The predicted protein effect of this variant: p. (Pro1432_Gly1433insAspTyrPheValGluIle). As a consequence, the stability of α5(IV) secondary structure was impaired, probably leading to the unusual configuration of α345(IV). Discussion: Normally, splicing variant in COL4A5 gene can lead to phenotypes of XLAS, and the effect is associated with the extent of splicing. The patient reported here carried a c.4298-20T>A splicing variant in COL4A5 gene, and AS was highly suspected based on the pathology results. However, the patient did not manifest any ocular or ear abnormalities. We therefore present the c.4298-20T>A splicing variant in COL4A5 gene as likely-pathogenic splicing variant that leads to XLAS with mild phenotypes.