Characterization of the VPS13B splicing pattern in a Cohen Syndrome Patient harboring a large deletion in the VPS13B gene

Characterization of the VPS13B splicing pattern in a Cohen Syndrome Patient harboring a large deletion in the VPS13B gene Fabiana Fanelli1, Roberta Saltarelli1, Marco Fichera3,4, Corrado Romano3,4, Elena Barbieri1, Rita Barone2, Giosuè Annibalini1 1University of Urbino Carlo Bo, Department of Biomolecular Sciences, Urbino, Italy; 2University of Catania, Child Neurology and Psychiatry Unit, Department of Clinical and Experimental Medicine, Catania, Italy; 3 Oasi Research Institute-IRCCS, Unit of Rare Diseases and Neurodevelopmental Disorders, Catania, Italy; 4University of Catania, Department of Biomedical and Biotechnological Sciences Section of Clinical Biochemistry and Medical Genetics, Catania, Italy. The human VPS13B gene is localized on chromosome 8 (8q22.2) and comprises 62 exons. Alternative splicing of VPS13B generates different mRNA variants with predicted open reading frames (ORFs) ranging from 1,248 to 12,069 nucleotides (nt). The longest transcript (NM_017890.5; isoform 5) encodes a predicted 4,022-amino acid transmembrane protein that plays a critical role in Golgi assembly and membrane transport [1]. Mutation in VPS13B causes Cohen syndrome, a rare autosomal disorder characterized by multisystemic involvement and a broad spectrum of clinical manifestations [2]. Here, we analyzed the VPS13B splicing pattern in a patient with Cohen syndrome who harbors a homozygous ≈250 Kb deletion on chromosome 8, leading to the complete loss of exons 18-24 and partial truncation of exon 25. Using primers spanning exon 17 to exon 29, we demonstrated that the patient expressed an mRNA variant with a complete deletion of exons 18-25. This deletion introduced a premature termination codon in the exon 26, reducing the ORF to 2,538 nt. Subsequently, using real-time PCR and VPS13B isoform-specific primers, we found that isoform 1 (NM_152564.5), which includes the mutually exclusive exon 28b and has an ORF of 11,994 nt, was the most highly expressed mRNA variant in both control and patient-derived fibroblasts and PBMCs. However, the patient exhibited a marked (2-3 fold) reduction of VPS13B isoform 1 mRNA expression compared to control fibroblasts, likely due to nonsense-mediated mRNA decay (NMD). Furthermore, VPS13B isoform 5 (NM_017890.5), which contains the mutually exclusive exon 28, was the second most highly expressed VPS13B mRNA isoform in control fibroblasts and PBMCs but was barely detectable in patient-derived cells. The expression of the other two VPS13B isoforms, namely NM_015243.3 (isoform 3) and NM_18166.1 (isoform 4) were not expressed in control and patient-derived fibroblasts and PBMCs. Interestingly, patient-derived fibroblasts expressed a previously undescribed mRNA isoform that included both exons 28 and 28b. Since both exons lie downstream of the gene mutation in the patient, this alternative splicing event did not alter the ORF length of the mutated VPS13B. In conclusion, we described a novel VPS13B mutation in a patient with Cohen syndrome. The large DNA deletion in this patient resulted in decreased VPS13B mRNA levels and a partial alteration of the slicing pattern. Further analyses are needed to evaluate the impact of this VPS13B mutation on protein production, localization and function. References [1] Seifert W. et al., J Biol Chem. 2011; 286(43):37665-75 [2] Kolehmainen J. et al., Am J Hum Genet. 2003 Jun;72(6):1359-69