Paper3_Rabacchi_APOBspliceVariants

Title: In Vitro Functional Characterization of Splicing Variants of the APOB Gene in Familial Hypobetalipoproteinemia

Authors and Institutions

• Authors: Claudio Rabacchi, PhD; Maria Luisa Simone, PhD; Livia Pisciotta, MD; Enza Di Leo, PhD; Davide Bocchi, MD; Antonello Pietrangelo, MD; Sergio D’Addato, MD; Stefano Bertolini, MD; Sebastiano Calandra, MD; Patrizia Tarugi, PhD.

• Institutions:

  • Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy

  • Department of Internal Medicine, University of Genova, Genova, Italy

  • Division of Internal Medicine and Center for Hemochromatosis, University of Modena and Reggio Emilia, Modena, Italy

  • Atherosclerosis and Metabolic Disease Research Unit, Department of Medical and Surgical Sciences, University of Bologna, Bologna, Italy.

Keywords

• APOB gene, Familial hypobetalipoproteinemia, Splicing variants, Truncated apoBs.

Background

• Familial Hypobetalipoproteinemia Type 1 (FHBL-1):

  • A codominant disorder with significantly reduced plasma levels of total cholesterol, low-density lipoprotein cholesterol, and apolipoprotein B (ApoB).

  • Often associated with rare exonic pathogenic variants in the APOB gene, including nonsense variants, deletions, and nonsynonymous variants.

  • Intronic variants at intron/exon junctions affecting splicing are also noted, but their pathogenicity remains largely unestablished.

Objective

• To functionally characterize six splicing variants of the APOB gene identified in seven presumed FHBL-1 heterozygotes through in vitro techniques.

Methods

• ApoB Minigenes:

  • Each variant was expressed in COS-1 cells and their transcripts sequenced to determine splicing effects.

Results

• Variants Identified:

  • Four novel splice-site variants:

    • c.23711G.A, c.81815G.A, c.3000-1G.T, c.384211G.A.

    • All predicted to abolish splice site activity, generating abnormal transcripts through cryptic splice site activation or exon skipping.

    • All variants resulted in transcripts with a premature termination codon, leading to truncated and non-functional apoBs:

      • Predicted translation products:

        • c.23711G.A: p.(Lys41Serfs2) and p.(Val80Ilefs10)

        • c.81815G.A: p.(Asn274*)

        • c.3000-1G.T: p.(Leu1001Alafs*10)

        • c.384211G.A: p.(Ser1281Argfs*2).

  • Two rare variants:

    • c.905-15C.G and c.1618-4G.A had uncertain effects in silico but yielded only wild-type transcripts.

Conclusions

• The identified minigene expression studies provide evidence supporting the pathogenicity of four novel splice site variants of the APOB gene found in FHBL-1.

Introduction

• Familial hypobetalipoproteinemia type 1:

  • Markedly reduced plasma levels of total cholesterol, LDL-C, and ApoB linked to pathogenic variants in the APOB gene, which encodes functional forms of ApoB produced by the liver (ApoB-100) and intestine (ApoB-48).

  • Truncated forms of APOB are produced at lower rates, with secretion reduction correlating with the extent of truncation; hence, carriers may be asymptomatic or present with varying clinical manifestations such as fatty liver.

Study Design

• Informed consent was obtained from adult subjects and parents of minors, adhering to ethical standards in line with the Helsinki Declaration.

Methods

Lipid and Lipoprotein Analysis

• Plasma lipid levels measured post-overnight fast, including total cholesterol, triglycerides, HDL-C, and ApoB concentrations following standard laboratory protocols.

Gene Sequencing

• Genomic DNA isolated from peripheral blood; sequencing focused on major genes related to primary hypobetalipoproteinemia using Sanger and NGS technologies.

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Results

Proband Characteristics

• Seven index patients identified with hypobetalipoproteinemia from routine lab tests. Phenotypic relationships established with first-degree relatives. Clinical features recorded, including fatty liver and associated serum transaminase elevations.

Specific Findings on Splicing Variants

Specific Findings on Splicing Variants

• Proband HBL-318: The c.23711G.A variant is located at the donor splice site in intron 3. Its predicted effect is to significantly disrupt the normal splicing patterns of the APOB mRNA, potentially leading to the exclusion of essential exons, and resulting in improper protein synthesis, which may contribute to the clinical symptoms observed in patients with Familial Hypobetalipoproteinemia Type 1 (FHBL-1).

• Proband HBL-147: The c.81815G.A variant affects the donor splice site in intron 7 and, based on in silico predictive tools, it is expected to alter splice site activity. Such changes can create aberrant splicing outcomes, resulting in transcripts that may translate to truncated forms of ApoB, decreasing overall functionality of this protein in lipid metabolism.

• Proband HBL-205 and HBL-134: The c.905-15C.G variant demonstrated varied results concerning its splicing impact, indicating potential variability in effects based on different cellular contexts or due to the influence of other genetic factors. These findings highlight the complexity of predicting splice variant consequences and the need for functional studies to clarify their roles.

• Proband HBL-166: This proband presented with two rare variants, both of which were confirmed to have unclear impacts on splicing. Further functional analysis is necessary to investigate how these variants might influence splicing or whether they have any pathogenic role in FHBL-1.

• Proband HBL-293: The identified c.3000-1G.T variant impacts the acceptor splice site and is anticipated to disturb the regular splicing process. This alteration could lead to the skipping of adjacent exons or the use of cryptic splice sites, ultimately resulting in non-functional ApoB protein variants.

• Proband HBL-251: The c.384211G.A variant affects the donor splice site in intron 24, predicted to yield abnormal splice products. This intrusion into normal splicing mechanisms can result in the generation of truncated proteins or proteins with altered amino acid sequences, further exacerbating the lipid profile and symptoms associated with FHBL-1.

• Proband HBL-147: c.81815G.A variant affecting donor splice site in intron 7 showed changes in splice site activity according to in silico predictive tools.

• Proband HBL-205 and HBL-134: c.905-15C.G variant showed varied results regarding splicing impact.

• Proband HBL-166: Two rare variants confirmed with unclear splicing impact.

• Proband HBL-293: c.3000-1G.T variant affecting acceptor splice site was found.

• Proband HBL-251: c.384211G.A affecting donor splice of intron 24 yielded abnormal splice products.

Discussion

• Study underscores the relevance of investigating novel splice variants of the APOB gene in the context of FHBL-1 with emphasis on their impact on protein functionality.

• Functional analyses provided new insights into the mechanisms leading to hypobetalipoproteinemia linked to splicing events and revealed additional previously uncharacterized variants.

Limitations

• Characterization performed in a heterologous cell model may not fully replicate in vivo splicing dynamics in human hepatocytes or enterocytes.

Conclusions

• The minigene expression studies substantiate the assignment of pathogenicity to four novel splice-site variants of the APOB gene in subjects clinically diagnosed with FHBL-1.

Acknowledgments

• Contributions from various authors in clinical management, study design, and article writing noted. Financial backing from MIUR and University of Modena and Reggio Emilia acknowledged.