A novel mutation (c.121-13T&gt;A) in the polypyrimidine tract of the splice acceptor site of intron 2 causes exon 3 skipping in mitochondrial acetoacetyl-CoA thiolase gene

Aoyama, Yuka; Sasai, Hideo; Abdelkreem, Elsayed; Otsuka, Hiroki; Nakama, Mina; Kumar, Sandeep; Aroor, Shrikiran; Shukla, Anju; Fukao, Toshiyuki

doi:10.3892/mmr.2017.6434

Cited by 7 publications

(5 citation statements)

References 31 publications

(32 reference statements)

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“…Moreover, other substitutions in this position (T>A,C,G) also cause exon 3 skipping. Aoyama et al ( 2017 ) PTC premature termination codon, NMD nonsense-mediated decay …”

Section: Cis -Element Splicing Mutationsmentioning

confidence: 99%

Splicing mutations in human genetic disorders: examples, detection, and confirmation

Abramowicz¹,

Gos²

2018

J Appl Genetics

508

420

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Precise pre-mRNA splicing, essential for appropriate protein translation, depends on the presence of consensus “cis” sequences that define exon-intron boundaries and regulatory sequences recognized by splicing machinery. Point mutations at these consensus sequences can cause improper exon and intron recognition and may result in the formation of an aberrant transcript of the mutated gene. The splicing mutation may occur in both introns and exons and disrupt existing splice sites or splicing regulatory sequences (intronic and exonic splicing silencers and enhancers), create new ones, or activate the cryptic ones. Usually such mutations result in errors during the splicing process and may lead to improper intron removal and thus cause alterations of the open reading frame. Recent research has underlined the abundance and importance of splicing mutations in the etiology of inherited diseases. The application of modern techniques allowed to identify synonymous and nonsynonymous variants as well as deep intronic mutations that affected pre-mRNA splicing. The bioinformatic algorithms can be applied as a tool to assess the possible effect of the identified changes. However, it should be underlined that the results of such tests are only predictive, and the exact effect of the specific mutation should be verified in functional studies. This article summarizes the current knowledge about the “splicing mutations” and methods that help to identify such changes in clinical diagnosis.

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“…Moreover, other substitutions in this position (T>A,C,G) also cause exon 3 skipping. Aoyama et al ( 2017 ) PTC premature termination codon, NMD nonsense-mediated decay …”

Section: Cis -Element Splicing Mutationsmentioning

confidence: 99%

Splicing mutations in human genetic disorders: examples, detection, and confirmation

Abramowicz¹,

Gos²

2018

J Appl Genetics

508

420

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“…Nevertheless, two of these variants, c.121–13T>A and c.941–9T>A, are located at the polypyrimidine tract of the splice acceptor site. Although in silico tools failed to predict the pathogenic effect of the latter two variants on splicing, minigene splicing experiments recently proved that c.121–13T>A and c.941–9T>A variants induce skipping of exons 3 and 10, respectively, in greater than 90% of transcripts (Aoyama et al, ; Sasai et al, ).…”

Section: Disease‐associated Acat1 Variantsmentioning

confidence: 99%

Mutation update on ACAT1 variants associated with mitochondrial acetoacetyl‐CoA thiolase (T2) deficiency

et al. 2019

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Mitochondrial acetoacetyl‐CoA thiolase (T2, encoded by the ACAT1 gene) deficiency is an inherited disorder of ketone body and isoleucine metabolism. It typically manifests with episodic ketoacidosis. The presence of isoleucine‐derived metabolites is the key marker for biochemical diagnosis. To date, 105 ACAT1 variants have been reported in 149 T2‐deficient patients. The 56 disease‐associated missense ACAT1 variants have been mapped onto the crystal structure of T2. Almost all these missense variants concern residues that are completely or partially buried in the T2 structure. Such variants are expected to cause T2 deficiency by having lower in vivo T2 activity because of lower folding efficiency and/or stability. Expression and activity data of 30 disease‐associated missense ACAT1 variants have been measured by expressing them in human SV40‐transformed fibroblasts. Only two variants (p.Cys126Ser and p.Tyr219His) appear to have equal stability as wild‐type. For these variants, which are inactive, the side chains point into the active site. In patients with T2 deficiency, the genotype does not correlate with the clinical phenotype but exerts a considerable effect on the biochemical phenotype. This could be related to variable remaining residual T2 activity in vivo and has important clinical implications concerning disease management and newborn screening.

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“…Despite minor modifications of endogenous Gnao1 ( Figure 2D ), two single-base substitutions can potentially cause an imbalance in allele expression. In particular, intronic mutation c.593 + 762C>A is located within the splicing signal and can affect the pre-mRNA processing leading to skipping of exon 6 ( Aoyama et al, 2017 ). We did not detect transcripts with skipped exon 6 in Gnao1-GGA mice ( Figure 3D ).…”

Section: Discussionmentioning

confidence: 99%

CRISPR/Cas9-generated mouse model with humanizing single-base substitution in the Gnao1 for safety studies of RNA therapeutics

et al. 2023

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The development of personalized medicine for genetic diseases requires preclinical testing in the appropriate animal models. GNAO1 encephalopathy is a severe neurodevelopmental disorder caused by heterozygous de novo mutations in the GNAO1 gene. GNAO1 c.607 G>A is one of the most common pathogenic variants, and the mutant protein Gαo-G203R likely adversely affects neuronal signaling. As an innovative approach, sequence-specific RNA-based therapeutics such as antisense oligonucleotides or effectors of RNA interference are potentially applicable for selective suppression of the mutant GNAO1 transcript. While in vitro validation can be performed in patient-derived cells, a humanized mouse model to rule out the safety of RNA therapeutics is currently lacking. In the present work, we employed CRISPR/Cas9 technology to introduce a single-base substitution into exon 6 of the Gnao1 to replace the murine Gly203-coding triplet (GGG) with the codon used in the human gene (GGA). We verified that genome-editing did not interfere with the Gnao1 mRNA or Gαo protein synthesis and did not alter localization of the protein in the brain structures. The analysis of blastocysts revealed the off-target activity of the CRISPR/Cas9 complexes; however, no modifications of the predicted off-target sites were detected in the founder mouse. Histological staining confirmed the absence of abnormal changes in the brain of genome-edited mice. The created mouse model with the “humanized” fragment of the endogenous Gnao1 is suitable to rule out unintended targeting of the wild-type allele by RNA therapeutics directed at lowering GNAO1 c.607 G>A transcripts.

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A novel mutation (c.121-13T>A) in the polypyrimidine tract of the splice acceptor site of intron 2 causes exon 3 skipping in mitochondrial acetoacetyl-CoA thiolase gene

Cited by 7 publications

References 31 publications

Splicing mutations in human genetic disorders: examples, detection, and confirmation

Splicing mutations in human genetic disorders: examples, detection, and confirmation

Mutation update on ACAT1 variants associated with mitochondrial acetoacetyl‐CoA thiolase (T2) deficiency

CRISPR/Cas9-generated mouse model with humanizing single-base substitution in the Gnao1 for safety studies of RNA therapeutics

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