Strategy for robust detection of insertions, deletions, and point mutations in CEBPA, a GC-rich content gene, using 454 next-generation deep-sequencing technology

CEBPA mutations are of prognostic relevance in acute myeloid leukemia (AML) and are currently detected using a combination of denaturing high-performance liquid chromatography (DHPLC), gene scan/fragment length analysis, and direct Sanger sequencing. Next-generation deep pyrosequencing, principally, allows for the highly sensitive detection of molecular mutations. However, standard 454 chemistry laboratory procedures lack efficient amplification of guanine-cytosine (GC)-rich amplicons during the emulsion PCR (emPCR) steps allowing direct massively parallel clonal amplification of PCR products. To solve this problem, we investigated six distinct emPCR conditions. The coding sequence of CEBPA was subdivided into four overlapping amplicons: GC content for amplicon 1, 74%; amplicon 2, 76%; amplicon 3, 77%; and amplicon 4, 69%. A new emPCR condition, improving the standard titanium assay, presents a robust solution to sequence amplicons with a GC content of up to 77%. Moreover, this assay was subsequently tested on a larger independent cohort of 23 AML patients. For each patient, a median of 737 reads was generated (coverage range, 397-fold to 1194-fold) and therefore allowed a robust detection of insertions, deletions, and point mutations. In conclusion, next-generation amplicon sequencing enables the highly sensitive detection of molecular mutations and is a feasible assay for routine assessment of GC-rich content amplicons.     

Risk factors and consequences of excessive autonomic activation during sleep in children

Purpose

The aim of this study was to assess risk factors for excessive autonomic activation during sleep (EAAS) and its association with sleep problems, impaired behavior, and poor academic performance in primary school children.

Methods

Data from a community-based study on 997 primary school children were used. Based on nocturnal home pulse oximetry, autonomic activation during sleep was defined as a pulse rate increase by more than 20%. Children with ??35.9 autonomic activations per hour (i.e., ??the 95^th centile) were classified as suffering from EAAS and compared with controls. Sleep problems, impaired behavior, and academic performance were assessed by parental questionnaires and analysis of school reports.

Results

According to the abovementioned definition, EAAS was diagnosed in 52 children (67% male). Risk factors for EAAS were male gender (odds ratio [95% confidence interval]: 2.06 [1.14?C3.72]) and presence of symptoms of sleep-disordered breathing (3.48 [1.29?C9.43]). Children with EAAS had a higher prevalence of hyperactive behavior (39.2% vs. 26.0%; p?=?0.05) and enuresis (5.8% vs. 0.8%; p?=?0.017) but not of poor academic performance. The association with hyperactive behavior was confirmed in a subsample (n?=?119) using the Strengths and Difficulties Questionnaire. Mean (SD) score of the hyperactive?Cinattentive scale was 4.5 (2.8) for EAAS and 3.4 (2.7) for non-EAAS (p?=?0.04).

Conclusion

EAAS may be a marker of sleep disruption in children and may predict the occurrence of enuresis and hyperactive behavior.     

Extracellular matrix expression of human tenocytes in three‐dimensional air–liquid and PLGA cultures compared with tendon tissue: Implications for tendon tissue engineering

Tenocyte transplantation may prove to be an approach to support healing of tendon defects. Cell–cell and cell–matrix contacts within three‐dimensional (3D) cultures may prevent tenocyte dedifferentiation observed in monolayer (2D) culture. The present study compares both neotissue formation and tenocyte extracellular matrix (ECM) expression in 2D and 3D cultures directly with that of native tendon, in order to determine optimal conditions for tendon tissue engineering. Primary human tenocytes were embedded in poly[lactic‐co‐glycolic‐acid] (PLGA)‐scaffolds and high‐density cultures. Neotissue formation was examined by hematoxyline–eosine (H&E) and immunofluorescence staining. Gene expression of ECM proteins and vascular endothelial growth factor (VEGF) was compared at days 0 (2D), 14, and 28 in 3D cultures and tendon. Histomorphology of 3D culture showed tendon‐like tissue as tenocyte cell nuclei became more elongated and ECM accumulated. Type I collagen gene expression was higher in 2D culture than in tendon and decreased in 4‐week‐old 3D cultures, whereas type III collagen was only elevated in high‐density culture compared with tendon. Decorin and COMP were reduced in 2D and increased in 3D culture almost to ex vivo level. These results suggest that the 3D high‐density or biodegradable scaffolds cultures encourage the differentiation of expanded monolayer tenocytes in vitro to tendon‐like tissue. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1170–1177, 2010