Cryptic splicing events in the iron transporter ABCB7 and other key target genes in SF3B1-mutant myelodysplastic syndromes.

The splicing factor SF3B1 is the most frequently mutated gene in myelodysplastic syndromes (MDS), and is strongly associated with the presence of ring sideroblasts (RS). We have performed a systematic analysis of cryptic splicing abnormalities from RNA sequencing data on hematopoietic stem cells (HSCs) of SF3B1-mutant MDS cases with RS. Aberrant splicing events in many downstream target genes were identified and cryptic 3' splice site usage was a frequent event in SF3B1-mutant MDS. The iron transporter ABCB7 is a well-recognized candidate gene showing marked downregulation in MDS with RS. Our analysis unveiled aberrant ABCB7 splicing, due to usage of an alternative 3' splice site in MDS patient samples, giving rise to a premature termination codon in the ABCB7 mRNA. Treatment of cultured SF3B1-mutant MDS erythroblasts and a CRISPR/Cas9-generated SF3B1-mutant cell line with the nonsense-mediated decay (NMD) inhibitor cycloheximide showed that the aberrantly spliced ABCB7 transcript is targeted by NMD. We describe cryptic splicing events in the HSCs of SF3B1-mutant MDS, and our data support a model in which NMD-induced downregulation of the iron exporter ABCB7 mRNA transcript resulting from aberrant splicing caused by mutant SF3B1 underlies the increased mitochondrial iron accumulation found in MDS patients with RS

Surfactant nebulization versus instillation during high frequency ventilation in surfactant-deficient rabbits

Surfactant nebulization improves lung function at low alveolar doses of surfactant. However, efficiency of nebulization is low, and lung deposition seems to depend on lung aeration. High frequency ventilation (HFV) has been shown to improve lung aeration. We hypothesize that the combination of HFV and surfactant nebulization may benefit lung deposition of surfactant and consequently, lung function. The aim of this study was to compare the effect of surfactant nebulization versus instillation during HFV on lung function, surfactant distribution, and cerebral blood flow. Therefore, severe respiratory failure was induced by lung lavages in 18 rabbits. HFV was applied: frequency = 8 Hz, mean airway pressure = 12 cm H2O, amplitude = 100%, fraction of inspired O-2 = 1.0. Technetium-99m-labeled surfactant (Alveofact, 100 mg/kg of BW) was nebulized or instilled (n = 6 each). Six other rabbits did not receive surfactant (control, HFV only). We found that after instillation partial arterial O-2 tension increased from 7.0 kPa (95% confidence interval, 6.3-8.0 kPa) to 34 kPa (16-51 kPa), and during nebulization from 7.0 kPa (6.0-9.0 kPa) to 46 kPa (27-58 kPa). Partial arterial CO, tension decreased after instillation from 6.1 kPa (5.3-7.1 kPa) to 4.8 kPa (3.9-5.6 kPa), and during nebulization, after an initial rise, it decreased from 6.3 kPa (5.3-7.4 kPa) to 4.9 kPa (4.4-5.6 kPa). Both treatments resulted in nonuniform distribution. Surfactant deposition after nebulization was 9.8%, Instillation resulted in a drop of mean arterial blood pressure of 17% (8-31%), and an even more pronounced drop in cerebral blood flow of 39% (18-57%). Nebulization did not affect blood pressure. Cerebral blood flow decreased with a maximum of 27% (10-37%). We conclude that surfactant nebulization during HFV improves lung function in rabbits with severe respiratory failure, without improving distribution, but with less effects on blood pressure and cerebral blood flow, when compared with surfactant instillation