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. 2024 Dec 19:12:1505924.
doi: 10.3389/fped.2024.1505924. eCollection 2024.

A novel variant c.7104 + 6T > A of ABCA12 linked to autosomal recessive congenital ichthyosis verified by minigene splicing assay

Linyan Zhu  1 , Rui Zhou  1 , Lianxiao Zhang  1 , Mei Chen  2 , Shengmin Zhang  2 , Xiaxi Huang  1 , Yubo Shi  1 , Huiqing Ding  1
Affiliations

A novel variant c.7104 + 6T > A of ABCA12 linked to autosomal recessive congenital ichthyosis verified by minigene splicing assay

Linyan Zhu et al. Front Pediatr. .

Abstract

Background: Autosomal recessive congenital ichthyosis (ARCI) is a group of genetic skin disorders characterized by abnormal keratinization, leading to significant health issues and reduced quality of life. ARCI encompasses harlequin ichthyosis (HI), congenital ichthyosiform erythroderma (CIE), and lamellar ichthyosis (LI). While all ARCI genes are linked to LI and CIE, HI is specifically associated with severe mutations in the ABCA12 gene. Milder forms like LI and CIE usually involve at least one non-truncating ABCA12 variant.

Methods: Whole-exome sequencing (WES) was performed on fetal and parental DNA, and ABCA12 gene variants were validated by Sanger sequencing. The functional effect of the novel variant c.7104 + 6T > A was evaluated using an in vitro minigene system, with splicing analysis conducted via PCR and Sanger sequencing.

Results: A compound heterozygous variation in the ABCA12 gene, comprising c.5784G > A (p.W1928*) and c.7104 + 6T > A, was identified in the fetus, inherited from the father and mother, respectively. According to ACMG guidelines, the c.7104 + 6T > A variant is classified as a Variant of Uncertain Significance (VUS). Computational predictions suggested that this variant affects splicing. A minigene assay further confirmed that the c.7104 + 6T > A variant in ABCA12 leads to two types of aberrant mRNA splicing: a 69-base pair deletion (c.7036_7104del, p.Val2346_Glu2368del) and skipping of Exon 47, both of which result in a premature stop codon and a truncated protein.

Conclusion: In conclusion, this study identified a novel genetic variant, c.7104 + 6T > A in ABCA12, as the cause of ARCI in a fetus, thereby enriched the known ABCA12 mutation spectrum.

Keywords: ABCA12 gene; autosomal recessive congenital ichthyosis (ARCI); mRNA splicing; minigene assay; variant.

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Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Clinical phenotype (A) the pedigree of the family. II2 (the proband) had two compound variants in ABCA12 gene, which are inherited from the I1(father) and the I2(mother), respectively. (B) Fetal ultrasound scan showed: eversion of the lips (eclabium), flexion of fingers. (C) Sanger sequencing analysis. The two variants (c.5784 G > A and c.7104 + 6 T > A) were validated by Sanger sequencing. (red arrows indicated the mutation).
Figure 2
Figure 2
Predictive results of the c.7104 + 6 T > A variant site in splicing. (A) Yellow arrow indicates location of the c.7104 + 6 T > A variant. (B) RDDCSC Predicts that c.7104 + 6 T > A variant of ABCA12 gene affects splicing by deleting 69 bp exon. (C) The SpliceAI algorithm shows that the original Acceptor site confidence score decreases by 0.39 after the mutation, the original Donor site confidence score decreases by 0.27, and a new Donor site confidence score of 0.21 may be generated, indicating that the c.7104 + 6 T > A variant may affect splicing. (D) The Predictive result shows that the original donor site is disrupted after the mutation, suggesting that the mutation may affect splicing by using FF.
Figure 3
Figure 3
Detection results of the pcDNA3.1 vector. (A) Minigene construction strategy diagram. (B) RT-PCR transcription analysis of agarose gel electrophoresis image in the pcDNA3.1 vector and splicing schematic diagram, the bands are labeled a, b, c in HeLa and 293 T cells. (C) Sequencing results corresponding to the splicing bands.
Figure 4
Figure 4
pcMINI-C vector detection results. (A) Minigene construction strategy diagram. (B) RT-PCR transcription analysis of agarose gel electrophoresis image in the pcMINI-C vector and splicing schematic diagram, the bands are labeled a, b, c in HeLa and 293 T cells. (C) Sequencing results corresponding to the splicing bands.
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Grants and funding

The author(s) declare financial support was received for the research, authorship, and/or publication of this article. This work was supported by the Medical and Health Plan of Zhejiang (grant number 2024KY1522, 2023KY1053) and Zhejiang Traditional Chinese Medicine Science and Technology Plan Project (grant number 2024ZL895).

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