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Analysis of institutional authors

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Minigene Splicing Assays and Long-Read Sequencing to Unravel Pathogenic Deep-Intronic Variants in PAX6 in Congenital Aniridia

Publicated to:INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES. 24 (2): 1562- - 2023-01-01 24(2), DOI: 10.3390/ijms24021562

Authors: Tamayo A; Núñez-Moreno G; Ruiz C; Plaisancie J; Damian A; Moya J; Chassaing N; Calvas P; Ayuso C; Minguez P; Corton M

Affiliations

CHU Toulouse, Hop Purpan, Ctr Reference Affect Rares Genet Ophtalmol CARGO, F-31000 Toulouse, France - Author
Inst Salud Carlos III, Ctr Biomed Network Res Rare Dis CIBERER, Madrid 28029, Spain - Author
Univ Alcala, Fac Med & Hlth Sci, Dept Surg Med & Social Sci, Sci & Technol Campus, Alcala De Henares 28871, Spain - Author
Univ Autonoma Madrid IIS FJD, Fdn Jimenez Diaz Univ Hosp, Bioinformat Unit, Inst Invest Sanitaria,UAM, Madrid 28240, Spain - Author
Univ Autonoma Madrid IIS FJD, Fdn Jimenez Diaz Univ Hosp, Dept Genet & Genom, Inst Invest Sanitaria,UAM, Madrid 28040, Spain - Author
Univ Toulouse 3, INSERM, U1214, F-31000 Toulouse, France - Author
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Abstract

PAX6 haploinsufficiency causes aniridia, a congenital eye disorder that involves the iris, and foveal hypoplasia. Comprehensive screening of the PAX6 locus, including the non-coding regions, by next-generation sequencing revealed four deep-intronic variants with potential effects on pre-RNA splicing. Nevertheless, without a functional analysis, their pathogenicity could not be established. We aimed to decipher their impact on the canonical PAX6 splicing using in vitro minigene splicing assays and nanopore-based long-read sequencing. Two multi-exonic PAX6 constructs were generated, and minigene assays were carried out. An aberrant splicing pattern was observed for two variants in intron 6, c.357+136G>A and c.357+334G>A. In both cases, several exonization events, such as pseudoexon inclusions and partial intronic retention, were observed due to the creation or activation of new/cryptic non-canonical splicing sites, including a shared intronic donor site. In contrast, two variants identified in intron 11, c.1032+170A>T and c.1033-275A>C, seemed not to affect splicing processes. We confirmed the high complexity of alternative splicing of PAX6 exon 6, which also involves unreported cryptic intronic sites. Our study highlights the importance of integrating functional studies into diagnostic algorithms to decipher the potential implication of non-coding variants, usually classified as variants of unknown significance, thus allowing variant reclassification to achieve a conclusive genetic diagnosis.

Keywords
congenital aniridiadeep-intronic variantsgenerationgeneticslong-read sequencingmessenger-rnaminigene splicing assaysminion nanopore sequencingnon-canonical splicing sitespatientpax6site mutationNon-canonical splicing sitesPax6Pluripotent stem-cells

Quality index

Bibliometric impact. Analysis of the contribution and dissemination channel

The work has been published in the journal INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES due to its progression and the good impact it has achieved in recent years, according to the agency WoS (JCR), it has become a reference in its field. In the year of publication of the work, 2023, it was in position 66/313, thus managing to position itself as a Q1 (Primer Cuartil), in the category Biochemistry & Molecular Biology.

From a relative perspective, and based on the normalized impact indicator calculated from the Field Citation Ratio (FCR) of the Dimensions source, it yields a value of: 2.73, which indicates that, compared to works in the same discipline and in the same year of publication, it ranks as a work cited above average. (source consulted: Dimensions May 2025)

Specifically, and according to different indexing agencies, this work has accumulated citations as of 2025-05-14, the following number of citations:

  • Scopus: 5
  • OpenCitations: 4
Impact and social visibility

From the perspective of influence or social adoption, and based on metrics associated with mentions and interactions provided by agencies specializing in calculating the so-called "Alternative or Social Metrics," we can highlight as of 2025-05-14:

  • The use, from an academic perspective evidenced by the Altmetric agency indicator referring to aggregations made by the personal bibliographic manager Mendeley, gives us a total of: 14.
  • The use of this contribution in bookmarks, code forks, additions to favorite lists for recurrent reading, as well as general views, indicates that someone is using the publication as a basis for their current work. This may be a notable indicator of future more formal and academic citations. This claim is supported by the result of the "Capture" indicator, which yields a total of: 14 (PlumX).

With a more dissemination-oriented intent and targeting more general audiences, we can observe other more global scores such as:

  • The Total Score from Altmetric: 1.25.
  • The number of mentions on the social network X (formerly Twitter): 2 (Altmetric).

It is essential to present evidence supporting full alignment with institutional principles and guidelines on Open Science and the Conservation and Dissemination of Intellectual Heritage. A clear example of this is:

  • The work has been submitted to a journal whose editorial policy allows open Open Access publication.
Leadership analysis of institutional authors

This work has been carried out with international collaboration, specifically with researchers from: France.

There is a significant leadership presence as some of the institution’s authors appear as the first or last signer, detailed as follows: First Author (Tamayo A) .

the author responsible for correspondence tasks has been Corton M.