Search

Duplex sequencing (DS) is an error-corrected next-generation sequencing method in which molecular barcodes informatically link PCR-copies back to their source DNA strands, enabling computational removal of errors in consensus sequences. The resulting background of less than one artifactual mutation per 10 7 nucleotides allows for direct detection of somatic mutations. TwinStrand Biosciences, Inc. has developed a DS-based mutagenesis assay to sample the rat genome, which can be applied to genetic toxicity testing. To evaluate this assay for early detection of mutagenesis, a time-course study was conducted using male Hsd:Sprague Dawley SD rats (3 per group) administered a single dose of 40 mg/kg N-ethyl-N-nitrosourea (ENU) via gavage, with mutation frequency (MF) and spectrum analyzed in stomach, bone marrow, blood, and liver tissues at 3 h, 24 h, 7 d, and 28 d post-exposure. Significant increases in MF were observed in ENU-exposed rats as early as 24 h for stomach (site of contact) and bone marrow (a highly proliferative tissue) and at 7 d for liver and blood. The canonical, mutational signature of ENU was established by 7 d post-exposure in all four tissues. Interlaboratory analysis of a subset of samples from different tissues and time points demonstrated remarkable reproducibility for both MF and spectrum. These results demonstrate that MF and spectrum can be evaluated successfully by directly sequencing targeted regions of DNA obtained from various tissues⁠, a considerable advancement compared to currently used in vivo gene mutation assays., Competing Interests: Declaration of Competing Interest G.A.P., F.Y.L, J.E.H., E.K.S., L.N.W., D.N., C.C.V., and J.J.S. are employees (or former employees) and equity holders of TwinStrand Biosciences, Inc. F.Y.L, E.K.S., L.N.W., C.C.V., and J.J.S. are authors on one or more duplex sequencing-related patents., (Published by Elsevier B.V.)

Purpose: To assess the safety and clinical outcomes of 6-month treatment with testosterone gel 1% therapy in adolescent boys with primary hypogonadism resulting from Klinefelter syndrome (KS) or anorchia., Methods: This was a subgroup analysis of a multicenter, open-label study of adolescent boys (N = 86) with delayed puberty who received .5-5.0 g testosterone gel 1% daily for ≤6 months. Adolescent boys 12-17 years of age with KS (n = 21) or anorchia (n = 8), bone age ≥10.5 years, and baseline growth data ≥6 months were included in this analysis. Serum hormone levels (total/free testosterone, luteinizing hormone, dihydrotestosterone, follicle-stimulating hormone, and estradiol) were measured using validated assays. Safety was assessed through adverse events (AEs)., Results: At baseline, patients with KS were taller, weighed more, and had higher total testosterone levels (mean 174 vs. 19 ng/dL) than patients with anorchia. At 6 months, total and free testosterone and dihydrotestosterone levels increased 1.8- to 2.3-fold in the KS group and eight- to 10-fold in anorchia patients. Estradiol levels increased 1.9-fold in the anorchia group and 1.4-fold in the KS group after treatment. No clinically significant changes were noted for luteinizing hormone, follicle-stimulating hormone, and sex hormone-binding globulin concentrations in either group. Cough was the most common AE (eight of 29), followed by acne and headache (both four of 29). One anorchia and two KS patients discontinued prematurely., Conclusions: Once-daily testosterone gel application increased serum testosterone levels into the pubertal range and maintained pubertal testosterone levels during 6-month treatment. In this study, testosterone gel 1% raised testosterone levels and was associated with cough as the most common AE., (Copyright © 2014 Society for Adolescent Health and Medicine. Published by Elsevier Inc. All rights reserved.)