Last Nucleotide Substitutions of COL4A5 Exons Cause Aberrant Splicing

Aoto, Yuya; Horinouchi, Tomoko; Yamamura, Takehira; Kondo, Atsushi; Nagai, Sonoko; Ishiko, Shinya; Okada, Eri; Rossanti, Rini; Sakakibara, Noburu; Nagano, China; Awano, Hiroyuki; Nagase, Hiroaki; Shima, Yuko; Nakanishi, Koichi; Matsuo, Masafumi; Iijima, Kazumoto; Nozu, Kandai

doi:10.1016/j.ekir.2021.10.012

Cited by 17 publications

(24 citation statements)

References 46 publications

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“…It was confirmed by the minigene assay that the COL4A3 missense variant p. (Leu1598Arg) and synonymous variant p. (Thr255Thr) theoretically speculated based on the nucleotide substitutions at the genomic DNA level may be splicing variants. Similar phenomena had been observed in COL4A5 gene using in vitro minigene analysis combined with or without analysis of patient-derived RNA from urine sediments and peripheral blood leukocytes ( 9 , 11 ). These pathogenic variants cannot be accurately evaluated based on the NGS results of genomic DNA alone if mRNA analysis is not performed.…”

Section: Discussionsupporting

confidence: 71%

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Presumed COL4A3/COL4A4 Missense/Synonymous Variants Induce Aberrant Splicing

et al. 2022

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BackgroundThe incorrect interpretation of missense and synonymous variants can lead to improper molecular diagnosis and subsequent faulty genetic counselling. The aim of this study was to evaluate the pathogenicity of presumed COL4A3/COL4A4 missense and synonymous variants detected by next-generation sequencing to provide evidence for diagnosis and genetic counselling.MethodsPatients' clinical findings and genetic data were analysed retrospectively. An in vitro minigene assay was conducted to assess the effect of presumed COL4A3/COL4A4 missense and synonymous variants on RNA splicing.ResultsFive unclassified COL4A3/COL4A4 variants, which were detected in five of 343 patients with hereditary kidney diseases, were analysed. All of them were predicted to affect splicing by Human Splicing Finder. The presumed COL4A3 missense variant c.4793T > G [p. (Leu1598Arg)] resulted in a loss of alternative full-length transcript during the splicing process. The COL4A3 transcript carried synonymous variant c.765G > A [p. (Thr255Thr)], led to an in-frame deletion of exon 13. Nevertheless, variants c.3566G > A [p. (Gly1189Glu)] in COL4A3 and c.3990G > A [p. (Pro1330Pro)], c.4766C > T [p. (Pro1589Leu)] in COL4A4 exhibited no deleterious effect on splicing. Among the five patients harbouring the abovementioned COL4A3/COL4A4 variants, three patients were genetically diagnosed with autosomal recessive Alport syndrome, one patient was highly suspected of having thin basement membrane nephropathy, and the other patient was clinically diagnosed with Alport syndrome.ConclusionsCOL4A3 presumed missense variant p. (Leu1598Arg) and synonymous variant p. (Thr255Thr) affect RNA splicing, which highlights the prime importance of transcript analysis of unclassified exonic sequence variants for better molecular diagnosis and genetic counselling. Meanwhile, the reliability of splicing predictions by predictive tools for exonic substitutions needs to be improved.

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Section: Discussionsupporting

confidence: 71%

“…Splicing defects were determined according to agarose gel image patterns of the amplification products containing the wild-type and variant alleles. The criteria were based on those proposed by Aoto et al ( 9 ).…”

Section: Methodsmentioning

confidence: 99%

Presumed COL4A3/COL4A4 Missense/Synonymous Variants Induce Aberrant Splicing

et al. 2022

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“…Exonic nucleotide substitutions in COL4A5 close to canonical splice sites sometimes affect normal splicing, especially when the final nucleotide of an exon is affected 31 – 33 . To ensure that variants with unreported splicing effects were not included unintentionally, all variants within 3 bases of a canonical splice site were assessed using MaxEntScan 34 .…”

Section: Methodsmentioning

confidence: 99%

Pathogenicity of missense variants affecting the collagen IV α5 carboxy non-collagenous domain in X-linked Alport syndrome

Gibson

Sadeghi-Alavijeh

Gale

et al. 2022

Sci Rep

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X-linked Alport syndrome is a genetic kidney disease caused by pathogenic COL4A5 variants, but little is known of the consequences of missense variants affecting the NC1 domain of the corresponding collagen IV α5 chain. This study examined these variants in a normal (gnomAD) and other databases (LOVD, Clin Var and 100,000 Genomes Project) to determine their pathogenicity and clinical significance. Males with Cys substitutions in the collagen IV α5 NC1 domain reported in LOVD (n = 25) were examined for typical Alport features, including age at kidney failure. All NC1 variants in LOVD (n = 86) were then assessed for structural damage using an online computational tool, Missense3D. Variants in the ClinVar, gnomAD and 100,000 Genomes Project databases were also examined for structural effects. Predicted damage associated with NC1 substitutions was then correlated with the level of conservation of the affected residues. Cys substitutions in males were associated with the typical features of X-linked Alport syndrome, with a median age at kidney failure of 31 years. NC1 substitutions predicted to cause structural damage were overrepresented in LOVD (p < 0.001), and those affecting Cys residues or ‘buried’ Gly residues were more common than expected (both p < 0.001). Most NC1 substitutions in gnomAD (88%) were predicted to be structurally-neutral. Substitutions affecting conserved residues resulted in more structural damage than those affecting non-conserved residues (p < 0.001). Many pathogenic missense variants affecting the collagen IV α5 NC1 domain have their effect through molecular structural damage and 3D modelling is a useful tool in their assessment.

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“…However, no studies have addressed the characteristic of exonic variants in the COL4A5 gene which are likely to affect splicing. Therefore, we focused on the variants affecting the last nucleotide of exons in COL4A5 and conducted a comprehensive in vitro transcript analysis ( 22 ). We found 14 reported variants located in last nucleotide of any COL4A5 exon from the Human Gene Mutation Database (HGMD) and six novel variants from our cohort.…”

Section: Splicing Variants In Col4a5 Genementioning

confidence: 99%

“…As for intronic variants of COL4A5 , several studies report using the minigene assay including our studies ( 11 , 15 , 41 ). In addition, most recently, our group applied this in vitro splicing assay for variants located in exons of COL4A5 to clarify the characteristic of exonic splicing variants ( 22 , 24 ).…”

Section: Diagnostic Strategy Of Col4a5 Splicing Va...mentioning

confidence: 99%

The Contribution of COL4A5 Splicing Variants to the Pathogenesis of X-Linked Alport Syndrome

et al. 2022

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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.

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Last Nucleotide Substitutions of COL4A5 Exons Cause Aberrant Splicing

Cited by 17 publications

References 46 publications

Presumed COL4A3/COL4A4 Missense/Synonymous Variants Induce Aberrant Splicing

Presumed COL4A3/COL4A4 Missense/Synonymous Variants Induce Aberrant Splicing

Pathogenicity of missense variants affecting the collagen IV α5 carboxy non-collagenous domain in X-linked Alport syndrome

The Contribution of COL4A5 Splicing Variants to the Pathogenesis of X-Linked Alport Syndrome

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