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Refining clinically relevant parameters for mis-splicing risk in shortened introns with donor-to-branchpoint space constraint.

ABSTRACT: Intronic deletions that critically shorten donor-to-branchpoint (D-BP) distance of a precursor mRNA impose biophysical space constraint on assembly of the U1/U2 spliceosomal complex, leading to canonical splicing failure. Here we use a series of β-globin (HBB) gene constructs with intron 1 deletions to define D-BP lengths that present low/no risk of mis-splicing and lengths which are critically short and likely elicit clinically relevant mis-splicing. We extend our previous observation in EMD intron 5 of 46 nt as the minimal productive D-BP length, demonstrating spliceosome assembly constraint persists at D-BP lengths of 47-56 nt. We exploit the common HBB exon 1 β-thalassemia variant that strengthens a cryptic donor (NM_000518.5(HBB):c.79G > A) to provide a simple barometer for the earliest signs of space constraint, via cryptic donor activation. For clinical evaluation of intronic deletions, we assert D-BP lengths > 60 nt present low mis-splicing risk while space constraint increases exponentially with D-BP lengths < 55 nt, with critical risk and profound splicing abnormalities with D-BP lengths < 50 nt.

SUBMITTER: Zhang KY

PROVIDER: S-EPMC11291888 | biostudies-literature | 2024 Aug

REPOSITORIES: biostudies-literature

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Refining clinically relevant parameters for mis-splicing risk in shortened introns with donor-to-branchpoint space constraint.

Zhang Katharine Y KY Joshi Himanshu H Marchant Rhett G RG Bryen Samantha J SJ Dawes Ruebena R Yuen Michaela M Cooper Sandra T ST Evesson Frances J FJ

European journal of human genetics : EJHG 20240527 8

Intronic deletions that critically shorten donor-to-branchpoint (D-BP) distance of a precursor mRNA impose biophysical space constraint on assembly of the U1/U2 spliceosomal complex, leading to canonical splicing failure. Here we use a series of β-globin (HBB) gene constructs with intron 1 deletions to define D-BP lengths that present low/no risk of mis-splicing and lengths which are critically short and likely elicit clinically relevant mis-splicing. We extend our previous observation in EMD in ...[more]

PMID: 38802528

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Project description:BackgroundHeart transplantation (HTx) from brainstem dead (BSD) donors is the gold-standard therapy for severe/end-stage cardiac disease, but is limited by a global donor heart shortage. Consequently, innovative solutions to increase donor heart availability and utilisation are rapidly expanding. Clinically relevant preclinical models are essential for evaluating interventions for human translation, yet few exist that accurately mimic all key HTx components, incorporating injuries beginning in the donor, through to the recipient. To enable future assessment of novel perfusion technologies in our research program, we thus aimed to develop a clinically relevant sheep model of HTx following 24 h of donor BSD.MethodsBSD donors (vs. sham neurological injury, 4/group) were hemodynamically supported and monitored for 24 h, followed by heart preservation with cold static storage. Bicaval orthotopic HTx was performed in matched recipients, who were weaned from cardiopulmonary bypass (CPB), and monitored for 6 h. Donor and recipient blood were assayed for inflammatory and cardiac injury markers, and cardiac function was assessed using echocardiography. Repeated measurements between the two different groups during the study observation period were assessed by mixed ANOVA for repeated measures.ResultsBrainstem death caused an immediate catecholaminergic hemodynamic response (mean arterial pressure, p = 0.09), systemic inflammation (IL-6 - p = 0.025, IL-8 - p = 0.002) and cardiac injury (cardiac troponin I, p = 0.048), requiring vasopressor support (vasopressor dependency index, VDI, p = 0.023), with normalisation of biomarkers and physiology over 24 h. All hearts were weaned from CPB and monitored for 6 h post-HTx, except one (sham) recipient that died 2 h post-HTx. Hemodynamic (VDI - p = 0.592, heart rate - p = 0.747) and metabolic (blood lactate, p = 0.546) parameters post-HTx were comparable between groups, despite the observed physiological perturbations that occurred during donor BSD. All p values denote interaction among groups and time in the ANOVA for repeated measures.ConclusionsWe have successfully developed an ovine HTx model following 24 h of donor BSD. After 6 h of critical care management post-HTx, there were no differences between groups, despite evident hemodynamic perturbations, systemic inflammation, and cardiac injury observed during donor BSD. This preclinical model provides a platform for critical assessment of injury development pre- and post-HTx, and novel therapeutic evaluation.

Dataset Information

Refining clinically relevant parameters for mis-splicing risk in shortened introns with donor-to-branchpoint space constraint.

Publications

Refining clinically relevant parameters for mis-splicing risk in shortened introns with donor-to-branchpoint space constraint.

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