RESUMEN
Introduction: In mitochondrial DNA (mtDNA) depletion syndrome (MDS), patients cannot maintain sufficient mtDNA for their energy needs. MDS presentations range from infantile encephalopathy with hepatopathy (Alpers syndrome) to adult chronic progressive external ophthalmoplegia. Most are caused by nucleotide imbalance or by defects in the mtDNA replisome. There is currently no curative treatment available. Nucleoside therapy is a promising experimental treatment for TK2 deficiency, where patients are supplemented with exogenous deoxypyrimidines. We aimed to explore the benefits of nucleoside supplementation in POLG and TWNK deficient fibroblasts. Methods: We used high-content fluorescence microscopy with software-based image analysis to assay mtDNA content and membrane potential quantitatively, using vital dyes PicoGreen and MitoTracker Red CMXRos respectively. We tested the effect of 15 combinations (A, T, G, C, AT, AC, AG, CT, CG, GT, ATC, ATG, AGC, TGC, ATGC) of deoxynucleoside supplements on mtDNA content of fibroblasts derived from four patients with MDS (POLG1, POLG2, DGUOK, TWNK) in both a replicating (10% dialysed FCS) and quiescent (0.1% dialysed FCS) state. We used qPCR to measure mtDNA content of supplemented and non-supplemented fibroblasts following mtDNA depletion using 20 µM ddC and after 14- and 21-day recovery in a quiescent state. Results: Nucleoside treatments at 200 µM that significantly increased mtDNA content also significantly reduced the number of cells remaining in culture after 7 days of treatment, as well as mitochondrial membrane potential. These toxic effects were abolished by reducing the concentration of nucleosides to 50 µM. In POLG1 and TWNK cells the combination of ATGC treatment increased mtDNA content the most after 7 days in non-replicating cells. ATGC nucleoside combination significantly increased the rate of mtDNA recovery in quiescent POLG1 cells following mtDNA depletion by ddC. Conclusion: High-content imaging enabled us to link mtDNA copy number with key read-outs linked to patient wellbeing. Elevated G increased mtDNA copy number but severely impaired fibroblast growth, potentially by inhibiting purine synthesis and/or causing replication stress. Combinations of nucleosides ATGC, T, or TC, benefited growth of cells harbouring POLG mutations. These combinations, one of which reflects a commercially available preparation, could be explored further for treatment of POLG patients.
RESUMEN
Aim of the Study: Esophageal dilatations are commonly performed in pediatric patients who have undergone an esophageal atresia/tracheoesophageal fistula (EA/TEF) repair or following caustic injury. We sought to compare the practice of esophageal dilatation across different specialties. Methods: We analyzed all patients who had an esophageal dilatation at our center between April 2014 and December 2018. Patients were identified via prospectively maintained databases and clinical coding records. Patients had a combination of dilatations under each specialty: interventional radiology (IR), surgery, and gastroenterology. Results: Thirty-five individual patients underwent 226 dilatations, median dilatations per patient was 3 (1-40). The median age at first dilatation was 18 months (1-194 months). Sixty-eight percent of patients had a previous EA/TEF repair. IR performed 59% of dilatations, surgeons 26%, and 15% by gastroenterologists. Surgeons more frequently were performing initial dilatations (P < .05) and performed more dilatations in EA/TEF patients (P < .0001). There was a significant difference between the time from a surgical dilatation until the next dilatation, 3.7 months, compared with an IR dilatation, 1.8 months (ANOVA, P < .05). Surgeons more frequently increased the size of balloon used (57% versus 33% versus 39%, P < .01). There was no significant difference in balloon size between specialties or in the incremental increase in size between subsequent dilatations. There was one postprocedure perforation, managed conservatively (complication rate = 0.4%). Conclusion: We have demonstrated that on average, patients wait longer after a surgical dilatation until their next procedure, and surgical teams are more likely to increase the size of the dilating balloon. Surgeons tend to be more involved in their postoperative patients in the initial phases of stricture management. Our results suggest the feasibility and safety of a multispecialty approach for these patients.
