Progress Update: Understanding the initiation and progression of FSHD-1

by Anna Pakula
Note that a final and more comprehensive update will be sent once the data has been fully analyzed.

The goal of our studies was to characterize the gene expression levels in DUX4 (+) vs. DUX4 (-) human muscle cells to deepen the understanding of the initiation and progression of FSHD-1. We initially performed single cell sequencing of myoblasts cultivated in differentiation medium up to day 4 in June 2018, for 4 FSHD-1 and 4 Control samples. Each library consisted of ~3000 cells. For selected samples known to express DUX4 at higher level we sequenced 6000 cells. However, several samples had low representation in the multiplexed libraries. To avoid biases in analysis due to skewed sample representation, selected backup libraries were sequenced in a second batch, in December 2018, and these displayed good balance between the multiplexed libraries.

During this time, Van den Heuvel et al. (Hum Mol Genet, 2019) published the results of a study quite similar to ours. We have therefore adjusted the scope of our analysis to include a comparison to their study, including an overview of findings that are confirmed by our data, and an exploration of reasons for discrepancies. Some discrepancies may be technical in origin, for example due to the use of different scRNA-seq platforms (10x Chromium in Van den Heuval et al. vs. inDrops for us), different read lengths (114bp vs. 61bp), or different computational pipelines (Cell Ranger/Monocle vs. dropEst/Seurat).

Other discrepancies may be biological in origin, for example due to differences in individual cell-lines, in the muscle types that cells were derived from, or in culture conditions. We noticed that our cells did not display as high levels of MYH3 (a marker of late stage differentiation) as those in Van den Heuvel et al., despite being differentiated longer (4 days vs 3.5 days). We think that this is because our cells were plated at 50% confluency with no cell-cell contact, whereas cells in Van den Heuvel et al. were plated at higher confluency, using EDTA to avoid cell fusion, allowing them to reach later stages of differentiation with higher DUX4 expression. As in Van den Heuval et al., we detected DUX4 expression in a small number of FSHD cells and known DUX4 targets in a larger collection of FSHD cells, but at lower frequencies; our overall signal for DUX4 target expression e.g. in Principal Component Analysis was also milder. The analysis of our data is still in progress, and one goal is to see what insights can be gained from the expression of DUX4 in cells at earlier stages of differentiation.

Since we have leftover funds from the Amis FSH in France, we would like to sequence more cells using a transcript-specific library preparation; by amplifying with suitably designed nested primers this should give us better sensitivity to detect DUX4 and selected target transcripts in our existing backup libraries. These samples will be prepared at the Single Cell Transcriptomics Core at Harvard University in collaboration with the Broad Institute. In this way we hope to get more insight into the gene expression patterns of DUX4 expressing cells.