Validation of CFTR intronic variants identified during cystic fibrosis population screening by a minigene splicing assay

Giorgi, Gianpietro; Casarin, Alberto; Trevisson, Eva; Donà, Marta; Cassina, Matteo; Graziano, Claudio; Picci, Luigi; Clementi, Maurizio; Salviati, Leonardo

doi:10.1515/cclm-2014-1047

Cited by 12 publications

(6 citation statements)

References 14 publications

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“…12 As no patient's RNA was available, to confirm its pathogenicity, we functionally characterized the synonymous variant using a β-globin hybrid minigene assay, which has been effectively used to validate potential splicing variants (Figure 2c). 13 RT-PCR analysis from cells transfected with the mutant construct showed an aberrant splicing compared with controls and yielded two bands (Figure 2d): sequencing of the lower fragment revealed that it corresponds to shorter transcripts owing to the activation of a novel canonical AG/GT 5′ donor splice site within SF3B4 exon 3 and the use of two different AG acceptor sites (the first in position c.536_537, the second three nucleotides downstream) (Figure 2e).…”

Section: Resultsmentioning

confidence: 99%

A synonymous splicing mutation in the SF3B4 gene segregates in a family with highly variable Nager syndrome

et al. 2016

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Nager syndrome is a rare preaxial acrofacial dysostosis that is caused by heterozygous loss-of-function variants in SF3B4. This gene encodes for a protein required for the assembly of spliceosomal complexes, being a master gene for splicing regulation. The main clinical features of Nager syndrome include facial-mandibular and preaxial limb malformations, with normal cognitive functioning. Most Nager patients are sporadic, but few familial cases with a highly variable phenotype have been reported. In this work, we report a novel synonymous variant within exon 3 of the SF3B4 gene in a family with three members affected by Nager syndrome. No pathogenic variants have been detected in other 24 genes associated with syndromes characterized by mandibulo-facial anomalies. The pathogenicity of the mutation was demonstrated through a hybrid minigene assay, which confirmed an aberrant splicing with the creation of a cryptic splice site, and showed that this allele is hypomorphic. Our findings emphasize the importance to perform functional analyses to assess the possible consequences of synonymous variants and confirmed that hybrid minigenes represent an effective tool to evaluate the effects of variants on splicing, particularly when RNA is not available.

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Section: Resultsmentioning

confidence: 99%

A synonymous splicing mutation in the SF3B4 gene segregates in a family with highly variable Nager syndrome

et al. 2016

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“…For instance, genetic variants mapping to exons of a lincRNA may alter the lincRNA secondary structure, which can be partly predicted using RNAfold web server (Hofacker and Stadler, 2006). The 5 UTR (un-translated region) variants may affect the process of splicing and stability of RNA conformation, a functional splicing reporter minigene assay should be used to assess the effect of genetic variants on RNA splicing (Giorgi et al, 2015). Through the aforementioned knowable strategies, comprehensive functional verification of non-coding variants is very important to understand their biological consequence; there is an urgent need to explore more practical methods and strategies for functional verification research.…”

Section: Function Verificationmentioning

confidence: 99%

Significance of Single-Nucleotide Variants in Long Intergenic Non-protein Coding RNAs

Zou

Tan

et al. 2020

Front. Cell Dev. Biol.

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Single-nucleotide variants (SNVs) are the most common genetic variants and universally present in the human genome. Genome-wide association studies (GWASs) have identified a great number of disease or trait-associated variants, many of which are located in non-coding regions. Long intergenic non-protein coding RNAs (lincRNAs) are the major subtype of long non-coding RNAs; lincRNAs play crucial roles in various disorders and cellular models via multiple mechanisms. With rapid growth in the number of the identified lincRNAs and genetic variants, there is great demand for an investigation of SNVs in lincRNAs. Hence, in this article, we mainly summarize the significant role of SNVs within human lincRNA regions. Some pivotal variants may serve as risk factors for the development of various disorders, especially cancer. They may also act as important regulatory signatures involved in the modulation of lincRNAs in a tissue-or disorder-specific manner. An increasing number of researches indicate that lincRNA variants would potentially provide additional options for genetic testing and disease risk assessment in the personalized medicine era.

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“…Mis-regulation of splicing is associated with an increasing number of human pathologies, including neurodegenerative disorders, cancer and genetic diseases [ 13 ]. The minigene approach is able to detect mRNA splicing aberrations using cells that do not express the endogenous mRNA and is a powerful and simple molecular tool to analyze mutations that might affect splicing [ 14 , 15 , 16 , 17 ]. Here, we report two Chinese patients suffering from seizures who were found to have DHP deficiency caused by novel heterozygous DPYS mutations.…”

Section: Introductionmentioning

confidence: 99%

Altered Pre-mRNA Splicing Caused by a Novel Intronic Mutation c.1443+5G>A in the Dihydropyrimidinase (DPYS) Gene

Nakajima

Meijer

Zhang³

et al. 2016

IJMS

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Dihydropyrimidinase (DHP) deficiency is an autosomal recessive disease caused by mutations in the DPYS gene. Patients present with highly elevated levels of dihydrouracil and dihydrothymine in their urine, blood and cerebrospinal fluid. The analysis of the effect of mutations in DPYS on pre-mRNA splicing is hampered by the fact that DHP is primarily expressed in liver and kidney cells. The minigene approach can detect mRNA splicing aberrations using cells that do not express the endogenous mRNA. We have used a minigene-based approach to analyze the effects of a presumptive pre-mRNA splicing mutation in two newly identified Chinese pediatric patients with DHP deficiency. Mutation analysis of DPYS showed that both patients were compound heterozygous for a novel intronic mutation c.1443+5G>A in intron 8 and a previously described missense mutation c.1001A>G (p.Q334R) in exon 6. Wild-type and the mutated minigene constructs, containing exons 7, 8 and 9 of DPYS, yielded different splicing products after expression in HEK293 cells. The c.1443+5G>A mutation resulted in altered pre-mRNA splicing of the DPYS minigene construct with full skipping of exon 8. Analysis of the DHP crystal structure showed that the deletion of exon 8 severely affects folding, stability and homooligomerization of the enzyme as well as disruption of the catalytic site. Thus, the analysis suggests that the c.1443+5G>A mutation results in aberrant splicing of the pre-mRNA encoding DHP, underlying the DHP deficiency in two unrelated Chinese patients.

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Validation of CFTR intronic variants identified during cystic fibrosis population screening by a minigene splicing assay

Abstract: Hybrid minigenes assay are a simple and rapid tool to evaluate the effects of intronic variants without the need of analyzing patient's mRNA, and are particularly suited to analyze variants identified during population screenings.

Cited by 12 publications

References 14 publications

A synonymous splicing mutation in the SF3B4 gene segregates in a family with highly variable Nager syndrome

A synonymous splicing mutation in the SF3B4 gene segregates in a family with highly variable Nager syndrome

Significance of Single-Nucleotide Variants in Long Intergenic Non-protein Coding RNAs

Altered Pre-mRNA Splicing Caused by a Novel Intronic Mutation c.1443+5G>A in the Dihydropyrimidinase (DPYS) Gene

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