Determination of the pathogenicity of known COL4A5 intronic variants by in vitro splicing assay

Horinouchi, Tomoko; Nozu, Kandai; Yamamura, Takehira; Minamikawa, Shogo; Nagano, China; Sakakibara, Noburu; Nakanishi, Koichi; Shima, Yuko; Morisada, Naoya; Ishiko, Shinya; Aoto, Yuya; Nagase, Hiroaki; Takeda, Hiroyuki; Rossanti, Rini; Kono, Hiroshi; Matsuo, Masafumi; Iijima, Kazumoto

doi:10.1038/s41598-019-48990-9

Cited by 16 publications

(24 citation statements)

References 35 publications

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“…RNA splicing is a process involving the removal of the intervening, noncoding sequences of genes (introns) from pre-mRNA and the joining of the protein-coding sequences (exons) together, to enable translation of mRNA into a protein. Recent research has underlined the abundance and importance of splicing variants in the etiology of inherited diseases [ 7 – 11 ]. The most effective method to determine whether the selected variants affect splicing is to analyze the mRNA extracted from the relevant tissue of patients.…”

Section: Introductionmentioning

confidence: 99%

Functional analysis of suspected splicing variants in CLCN5 gene in Dent disease 1

et al. 2020

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Background In recent years, the elucidation of splicing abnormalities as a cause of hereditary diseases has progressed. However, there are no comprehensive reports of suspected splicing variants in the CLCN5 gene in Dent disease cases. We reproduced gene mutations by mutagenesis, inserted the mutated genes into minigene vectors, and investigated the pathogenicity and onset mechanisms of these variants. Methods We conducted functional splicing assays using a hybrid minigene for six suspected splicing variants (c.105G>A, c.105+5G>C, c.106−17T>G, c.393+4A>G, c.517−8A>G, c.517−3C>A) in CLCN5. We extracted information on these variants from the Human Gene Mutation Database. We reproduced minigene vectors with the insertion of relevant exons with suspected splicing variants. We then transfected these minigene vectors into cultured cells and extracted and analyzed the mRNA. In addition, we conducted in silico analysis to confirm our minigene assay results. Results We successfully determined that five of these six variants are pathogenic via the production of splicing abnormalities. One showed only normal transcript production and was thus suspected of not being pathogenic (c.106−17T>G). Conclusion We found that five CLCN5 variants disrupted the original splice site, resulting in aberrant splicing. It is sometimes difficult to obtain mRNA from patient samples because of the fragility of mRNA or its low expression level in peripheral leukocytes. Our in vitro system can be used as an alternative to in vivo assays to determine the pathogenicity of suspected splicing variants.

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Section: Introductionmentioning

confidence: 99%

Functional analysis of suspected splicing variants in CLCN5 gene in Dent disease 1

et al. 2020

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“…During such study, we have produced a ready-made splicing reporter minigene named H492 minigene [34]. This minigene has been utilized not only for analysis of exon skippable agents [11,19] but also for in vitro determination of splicing outcomes caused by nucleotide changes in genetic diseases [35][36][37][38][39][40][41][42]. However, the minigene is facing two drawbacks of RT-PCR amplification and sequencing step.…”

Section: Discussionmentioning

confidence: 99%

Dual Fluorescence Splicing Reporter Minigene Identifies an Antisense Oligonucleotide to Skip Exon v8 of the CD44 Gene

Fukushima

Farea

Maeta

et al. 2020

IJMS

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Splicing reporter minigenes are used in cell-based in vitro splicing studies. Exon skippable antisense oligonucleotide (ASO) has been identified using minigene splicing assays, but these assays include a time- and cost-consuming step of reverse transcription PCR amplification. To make in vitro splicing assay easier, a ready-made minigene (FMv2) amenable to quantitative splicing analysis by fluorescence microscopy was constructed. FMv2 was designed to encode two fluorescence proteins namely, mCherry, a transfection marker and split eGFP, a marker of splicing reaction. The split eGFP was intervened by an artificial intron containing a multicloning site sequence. Expectedly, FMv2 transfected HeLa cells produced not only red mCherry but also green eGFP signals. Transfection of FMv2CD44v8, a modified clone of FMv2 carrying an insertion of CD44 exon v8 in the multicloning site, that was applied to screen exon v8 skippable ASO, produced only red signals. Among seven different ASOs tested against exon v8, ASO#14 produced the highest index of green signal positive cells. Hence, ASO#14 was the most efficient exon v8 skippable ASO. Notably, the well containing ASO#14 was clearly identified among the 96 wells containing randomly added ASOs, enabling high throughput screening. A ready-made FMv2 is expected to contribute to identify exon skippable ASOs.

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“…Recently, we and other groups developed an in vitro splicing assay using a minigene construct to detect aberrant splicing caused by variants in COL4A5 (Fig. 7) [16,[29][30][31]. This assay can easily detect the pathogenicity of single-base substitutions causing aberrant splic-…”

Section: In Vitro Splicing Assay For Detecting Splicing Abnormalitiesmentioning

confidence: 99%

“…www.krcp-ksn.org ing in exons and introns, such as synonymous variants or variants outside of the intronic consensus sequences [16,30,32,33]. Here we describe one example of such detection (Fig.…”

Section: Lam Dapimentioning

confidence: 99%

“…Recently, we and other groups developed an in vitro splicing assay using a minigene construct to detect aberrant splicing caused by variants in COL4A5 ( Fig. 7 ) [ 16 , 29 - 31 ]. This assay can easily detect the pathogenicity of single-base substitutions causing aberrant splicing in exons and introns, such as synonymous variants or variants outside of the intronic consensus sequences [ 16 , 30 , 32 , 33 ].…”

Section: In Vitro Splicing Assay For Detecting Splicing Abnomentioning

confidence: 99%

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Genetic background, recent advances in molecular biology, and development of novel therapy in Alport syndrome

Nozu

Takaoka

Kai

et al. 2020

Kidney Res Clin Pract

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Alport syndrome (AS) is a progressive inherited kidney disease characterized by hearing loss and ocular abnormalities. There are three forms of AS depending on inheritance mode: X-linked Alport syndrome (XLAS), autosomal recessive AS (ARAS), and autosomal dominant AS (ADAS). XLAS is caused by pathogenic variants in COL4A5, which encodes type IV collagen α5 chain, while ADAS and ARAS are caused by variants in COL4A3 or COL4A4, which encode type IV collagen α3 or α4 chain, respectively. In male XLAS or ARAS cases, end-stage kidney disease (ESKD) develops around a median age of 20 to 30 years old, while female XLAS or ADAS cases develop ESKD around a median age of 60 to 70 years old. The diagnosis of AS is dependent on either genetic or pathological findings. However, determining the pathogenicity of the variants detected by gene tests can be difficult. Recently, we applied the following molecular investigation tools to determine pathogenicity: 1) in silico and in vitro trimer formation assay of α345 chains to assess triple helix formation ability, 2) kidney organoids constructed from patients' induced pluripotent stem cells to identify α5 chain expression on the glomerular basement membrane, and 3) in vitro splicing assay to detect aberrant splicing to determine the pathogenicity of variants. In this review article, we discuss the genetic background and novel assays for determining the pathogenicity of variants. We also discuss the current treatment approaches and introduce exon skipping therapy as one potential treatment option.

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Determination of the pathogenicity of known COL4A5 intronic variants by in vitro splicing assay

Cited by 16 publications

References 35 publications

Functional analysis of suspected splicing variants in CLCN5 gene in Dent disease 1

Functional analysis of suspected splicing variants in CLCN5 gene in Dent disease 1

Dual Fluorescence Splicing Reporter Minigene Identifies an Antisense Oligonucleotide to Skip Exon v8 of the CD44 Gene

Genetic background, recent advances in molecular biology, and development of novel therapy in Alport syndrome

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