Culture Conditions Affect Expression of DUX4 in FSHD Myoblasts.

Sachchida Pandey,Yi-Wen Chen,Hunain Khawaja

doi:10.3390/molecules20058304

Abstract

Facioscapulohumeral muscular dystrophy (FSHD) is believed to be caused by aberrant expression of double homeobox 4 (DUX4) due to epigenetic changes of the D4Z4 region at chromosome 4q35. Detecting DUX4 is challenging due to its stochastic expression pattern and low transcription level. In this study, we examined different cDNA synthesis strategies and the sensitivity for DUX4 detection. In addition, we investigated the effects of dexamethasone and knockout serum replacement (KOSR) on DUX4 expression in culture. Our data showed that DUX4 was consistently detected in cDNA samples synthesized using Superscript III. The sensitivity of DUX4 detection was higher in the samples synthesized using oligo(dT) primers compared to random hexamers. Adding dexamethasone to the culture media significantly suppressed DUX4 expression in immortalized (1.3 fold, p < 0.01) and primary (4.7 fold, p < 0.01) FSHD myoblasts, respectively. Culture medium with KOSR increased DUX4 expression and the response is concentration dependent. The findings suggest that detection strategies and culture conditions should be carefully considered when studying DUX4 in cultured cells.

Highlights

Facioscapulohumeral muscular dystrophy (FSHD) is a dominant muscular dystrophy, with a prevalence of 1:20,000, and the third most common type of muscular dystrophy [1]
Our result showed that double homeobox 4 (DUX4) expression was detected in FSHD myoblasts when the cDNA was synthesized using Superscript III
Our data showed that higher DUX4 expression (1.7 fold, p < 0.01) was detected in cDNA samples synthesized using oligo(dT) primers compared to random hexamers (Figure 2)

Summary

Introduction

Facioscapulohumeral muscular dystrophy (FSHD) is a dominant muscular dystrophy, with a prevalence of 1:20,000, and the third most common type of muscular dystrophy [1]. The disease is characterized by a progressive weakness and atrophy of the facial, scapular, and humeral muscles followed by weakness of muscles of the lower extremities [2]. FSHD is sub-classified into FSHD1 and FSHD2 depending on the genetic causes. FSHD1 is genetically linked to contractions of the D4Z4 repeat array at chromosome 4q35 and affects approximately 95% of the patients. In FSHD1, the D4Z4 array is contracted from 11–150 repeat units in unaffected individuals to. 1–10 repeat units in the patients [3,4,5]. Each D4Z4 repeat contains a double homeobox protein 4

Objectives

Methods

Results

Conclusion