Intracellular readthrough of nonsense mutations by aminoglycosides in coagulation factor VII.

BACKGROUND: Nonsense mutations in coagulation factor (F) VII potentially cause a lethal hemorrhagic diathesis. Readthrough of nonsense mutations by aminoglycosides has been studied in a few human disease models with variable results. OBJECTIVES: We investigated the K316X and W364X FVII mutations, associated with intracranial hemorrhage, and their correction by aminoglycosides. The rare nonsense mutations in FVII represent favorite models to test this strategy, because even tiny increases in the amount of functional full-length protein in patients could ameliorate hemorrhagic phenotypes. RESULTS: A FVII-green fluorescent protein (GFP) chimaera provided us with a fluorescent model of FVII expression in living cells. Appreciable fluorescence in cells transfected with nonsense FVII-GFP mutants was detected upon geneticin treatment, thus demonstrating suppression of premature translation termination. To investigate the rescue of FVII function, nonsense variants of the native FVII without GFP (p316X-FVII and p364X-FVII) were transfected and found to secrete low amounts of FVII (approximately 1% of Wt-FVII activity), thus suggesting a spontaneous stop codon readthrough. Geneticin treatment of cells resulted in a significant and dose-dependent increase of secreted FVII molecules (p316X-FVII, 24 +/- 12 ng mL(-1), 3.6 +/- 0.8% of Wt-FVII activity; p364X-FVII, 26 +/- 10 ng mL(-1), 3.7+/-0.6%) characterized by reduced specific activity, thus indicating the synthesis of dysfunctional proteins. Similar results were observed with gentamicin, a commonly used aminoglycoside of potential interest for patient treatment. CONCLUSIONS: Our approach, extendable to other coagulation factors, represents an effective tool for a systematic study of the effects of aminoglycosides and neighboring sequences on nonsense codon readthrough. These results provide the rationale for a mutation-specific therapeutic approach in FVII deficiency.

Clonazepam in childhood anxiety disorders.

OBJECTIVE: There is evidence for the efficacy and safety of clonazepam (CZP) in adult anxiety disorders, but no formal studies to substantiate clinical reports of similar benefit in children with anxiety disorders. METHOD: In this double-blind pilot study, 15 children, aged 7 to 13 years, entered a randomly assigned, double-blind crossover trial of 4 weeks of CZP (up to 2 mg/day) and 4 weeks of placebo. RESULTS: Twelve children completed the trial. All but 1 had a diagnosis of separation anxiety disorder, and all but 2 had comorbid diagnoses. Nine children appeared to have moderate to significant clinical improvement, but statistical comparisons on several ratings failed to confirm a trend in favor of CZP. Side effects of drowsiness, irritability, and/or oppositional behavior were notable in 10 children in the CZP phase compared with 5 in the placebo phase. CONCLUSIONS: Clonazepam was believed to have clinical benefit for some children, but this was not confirmed statistically in this small sample. Problematic side effects of drowsiness and disinhibition were common and possibly were due to rapid titration.

Proliferation centers in chronic lymphocytic leukemia: correlation with cytogenetic and clinicobiological features in consecutive patients analyzed on tissue microarrays.

To better define the significance of proliferation centers (PCs), the morphological hallmark of chronic lymphocytic leukemia (CLL), lymph node biopsies taken from 183 patients were submitted to histopathologic and fluorescence in situ hybridization (FISH) studies using a 5-probe panel on tissue microarrays. Seventy-five cases (40.9%) with confluent PCs were classified as 'PCs-rich' and 108 cases (59.1%) with scattered PCs were classified as 'typical'. Complete FISH data were obtained in 101 cases (55.1%), 79 of which (78.2%) displayed at least one chromosomal aberration. The incidence of each aberration was: 13q- 36,7%, 14q32 translocations 30.8%, 11q- 24.7%, trisomy 12 19.5% and 17p- 15.6%. Five cases showed extra copies of the 14q32 region. The 'PCs-rich' group was associated with 17p-, 14q32/IgH translocation, +12, Ki-67>30%. The median survival from the time of tissue biopsy for PCs-rich and typical groups was 11 and 64 months, respectively (P=0.00001). The PCs-rich pattern was the only predictive factor of an inferior survival at multivariate analysis (P=0.022). These findings establish an association between cytogenetic profile and the amount of PC in CLL, and show that this histopathologic characteristic is of value for risk assessment in patients with clinically significant adenopathy.