A generic assay for the identification of splicing variants that induce nonsense-mediated decay in Pompe disease.

DNA variants affecting mRNA expression and processing in genetic diseases are often missed or poorly characterized. We previously reported a generic assay to identify variants that affect mRNA expression and splicing in Pompe disease, a monogenic disorder caused by deficiency of acid α-glucosidase (GAA). However, this assay could miss mRNA that is subjected to degradation. Here, we inhibited mRNA degradation using cycloheximide and performed unbiased splicing analysis of all GAA exons using exon flanking RT-PCR and exon internal RT-qPCR. In four patients that were suspected of harboring splicing variants but for which aberrant splicing could not be detected in normally growing cells, we detected a total of 10 novel splicing events in cells treated with cycloheximide. In addition, we found that sequences of GAA introns 6 and 12 were naturally included in a subset of transcripts from patients and healthy controls, indicating inefficient canonical splicing. Identification of aberrant splicing caused by the common Asian variant c.546G>T allowed the development of an antisense oligonucleotide that promoted canonical GAA pre-mRNA splicing and elevated GAA enzymatic activity. Our results indicate that this extended generic splicing assay allows the detection of aberrant splicing in cases of mRNA degradation to enable functional analysis of unknown splicing variants and the development of targeted treatment options.

Transfusion outcome for volume- and plasma-reduced platelet concentrates for pediatric patients.

BACKGROUND AND OBJECTIVES: Plasma reduction in platelet concentrate (PC) products has been reported to prevent large volume load and transfusion-related adverse reactions (TRARs). However, volume reduction might be associated with a poor transfusion response because of a deterioration in platelet (PLT) quality. Because PLT quality control and transfusion responses for recently washed PCs using PLT additive solutions are superior, we investigated the clinical safety and transfusion efficacy of volume-reduced washed PCs in pediatric patients. MATERIALS AND METHODS: We prepared a simplified resuspended PC product (RPC) as a washed PC. Regular RPC (R-RPC) included equivalent volumes of bicarbonate Ringer's solution and anticoagulant citrate dextrose solution A (BRS-A) as the resuspension solution. Half RPC (H-RPC) was prepared by adding a half volume of BRS-A. Twenty-four pediatric patients were scheduled for transfusions with R-RPC and H-RPC up to 4 times. R-RPC was transfused 42 times into 24 patients. H-RPC was transfused 41 times into 23 patients. RESULTS: Neither product was observed to cause TRARs. Although the calculated PLT recovery for H-RPC was significantly reduced, the posttransfusion corrected count increment (24 h) did not differ. Moreover, similar results were observed for vital signs during transfusion. CONCLUSION: Volume-reduced washed PC can be transfused without causing TRARs, differences in vital signs, or inferior transfusion responses. Volume-reduced washed PC also provides the advantages of shortened transfusion times and reduced volume loads. Although a standard technique for stable resuspension is necessary, volume-reduced washed PC may be a beneficial option for children, including neonates, or individuals with cardiovascular or renal problems.

Investigation of factors associated with allergic transfusion reaction due to platelet transfusion and the efficacy of platelets resuspended in BRS-A in adult patients.

BACKGROUND: Although allergic transfusion reactions (ATRs) resulting from platelet concentrate (PC) are a common adverse reaction, the mechanism underlying ATRs has not been fully elucidated. Plasma-replaced PC suspended in bicarbonate Ringer's solution and anticoagulant citrate dextrose solution A (RPC-B) is effective for preventing ATRs in children in Japan; however, there is not enough evidence in adult populations. STUDY DESIGN AND METHODS: We conducted a retrospective analysis focused on factors associated with ATRs developing from PC transfusions in adult patients in a single institution between 2015 and 2018. The clinical efficacy of RPC-B for adult patients was also analyzed. RESULTS: In total, 4,677 untreated regular PC products in plasma were transfused into 914 patients. ATRs developed in 65 patients (7.1%) treated with 92 PC products (2.0%). Multivariate analysis revealed that patients who were elderly, diagnosed with a non-hematological disease, and who received a transfusion of fresh-frozen plasma and red blood cell concentrate products together with PC products had lower frequencies of ATRs. Although 40 patients received 490 RPC-B transfusions, six ATRs (1.2%) were confirmed in five patients (12.5%). The ATR frequency was not significantly lower in the analysis of all patients; however, ATRs in patients with hematological diseases were lower in terms of both the patient and product numbers. Corrected count increments (24 hr) were also within an acceptable range in patients with hematological diseases. CONCLUSION: Several patient-specific factors may be associated with the development of ATRs from PC transfusion. Because RPC-B appears to efficiently prevent ATRs, even in adult patients, safe and efficient transfusions may be performed by using RPC-B preferentially depending on the patient's risk factors.