Progress Update: SLC34A2 as a Biomarker for FSHD

Progress update by Dr. Robert Bloch
See grant SLC34A2 as a Biomarker for FSHD

We’ve been progressing in our research for Friends of FSH Research, despite the severe delays imposed by the pandemic and the cut-backs it has imposed on our level of activity. In particular,

Overview (recap): We have shared published and preliminary evidence that indicates that the sodium, phosphate cotransporter protein, SLC34A2, is detected in frozen cross sections of human FSHD but not in control muscle (10-fold lower levels in controls) as well as in biopsies and serum from mice carrying xenografts we prepared from FSHD myogenic cells. We proposed to follow up on these original findings in two aims. In our first aim, we proposed to learn how unique the SLC34A2 protein is in these respects. SLC34A2 is only one of dozens or even hundreds of gene products that are expressed at high levels in FSHD tissue as a result of the higher expression of the DUX4 transcription factor. Can other proteins be probed as potential biomarkers of FSHD, and if so, are they as potentially useful as SLC34A2 as a biomarker? In our second aim, we proposed to determine if SLC34A2 or its peptides could be detected at significantly higher levels in the serum of FSHD patients than in control human serum.

Set-backs: As researchers everywhere, we have taken a severe hit from the pandemic. Our university has just recently moved from Phase 1 to Phase 2 of its Covid-19 recovery plan. Manpower is still limited to 50% maximum and to no more than 1 person every 150 sq ft of lab space. Despite this, we’ve been making good progress.

Results, Deliverables and Progress in their Completion

Aim (i): Uniqueness of SLC34A2: We have ruled out a number of DUX4 target gene products as potentially useful protein biomarkers of FSHD, at least in our xenograft model, where distinguishing the human proteins from endogenous murine proteins is essential. Indeed, of the dozen or so candidates we have assayed, only one can be detected by immunofluorescence in the FSHD grafts at higher levels than in control grafts. Antibodies to the other proteins we have assayed that are now commercially available cannot be used reliably in biomarker assays. We are following up on our observation on that one protein, as well as with SLC34A2.

Related studies have probed the reliability of our immunological assays for SLC34A2. Our results suggest that we can reliably detect SLC34A2 for subsequent quantitative assays in both tissue and serum.

We have also confirmed the reliability of our immunoassays for other FSHD tissues. So far, most of our assays have been done with biopsy material from mice engrafted with cell lines that were derived from individuals in the same family. We have more recently examined grafts made from cells which were derived from an individual who was chimeric for FSHD markers. We found that the same antibodies that recognize the FSHD myofibers in the former grafts also recognize the same FSHD fibers in the latter grafts.

Aim (ii): Detection of SLC34A2 in FSHD but not control human serum: We are collaborating with the laboratory of Dr. Jeffrey Statland, Kansas University Medical Center (KUMC), on this aim. Due to delays caused by the Covid pandemic, processing and approval of the MTAs between the University of Maryland, Baltimore, and KUMC, and the subsequent delays in the Statland lab’s ability to ship the samples to us, we just received the samples (10 biopsies and 10 serum samples from the same FSHD patients) 3 weeks ago. We have just begun assaying the serum samples.

Future Plans

We continue to test antibodies to other gene products that are upregulated as a result of DUX4 expression in FSHD tissue. Another dozen or so additional antibodies from commercial sources remain on our list for testing. We will also be collaborating with Dr. Sujatha Jagannathan (U CO, Boulder), who has proteomic evidence that the protein she has focused on may also be a useful protein biomarker for FSHD.

We should soon begin to have results of immunoblots of human serum samples from FSHD patients and immunofluorescence labeling of the corresponding muscle biopsies. We will need control sera and biopsies, including those from individuals with other neuromuscular disorders, to complete this aim. We are already in discussions with the Statland lab to obtain these additional samples.